An improved amplification and sequencing strategy for phylogenetic studies using the mitochondrial large subunit rRNA gene

Genome ◽  
1996 ◽  
Vol 39 (4) ◽  
pp. 793-797 ◽  
Author(s):  
Alex Parker ◽  
Irv Kornfield
Keyword(s):  
16S Rrna ◽  
Molecular Phylogenetics ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Variable Region ◽  
Rrna Gene ◽  
Molecular Systematic ◽  
Wide Range ◽  
Sequencing Strategy ◽  
Large Subunit Rrna

Numerous molecular systematic studies have employed variation in the mitochondrial large subunit (16s) rRNA gene to infer patterns of relationship among species and higher taxa. The primers most commonly employed in 16s rRNA amplification and sequencing bracket an approximately 600 bp portion of this gene. However, most of the informative variation occurs within a 200 bp subset of this segment. We describe a novel primer pair designed to amplify this variable region in a wide range of taxa, allowing broader application and considerable streamlining of data acquisition for studies using this gene. Key words : molecular phylogenetics, polymerase chain reaction, mtDNA, large ribosomal subunit, control region.

scholarly journals Assessment of Diversity of Culturable Marine Yeasts Associated with Corals and Zoanthids in the Gulf of Thailand, South China Sea

2020 ◽  
Vol 8 (4) ◽  
pp. 474 ◽  
Author(s):  
Chutima Kaewkrajay ◽  
Thanongsak Chanmethakul ◽  
Savitree Limtong
Keyword(s):  
Marine Invertebrates ◽  
Large Subunit ◽  
Rrna Gene ◽  
Gulf Of Thailand ◽  
Marine Yeasts ◽  
D2 Domain ◽  
Wide Range ◽  
The Gulf Of Thailand ◽  
Ascomycetous Yeasts ◽  
Large Subunit Rrna

Marine yeasts can occur in a wide range of habitats, including in marine invertebrates, in which they may play important roles; however, investigation of marine yeasts in marine invertebrates is scarce. Therefore, this study aims to explore the diversity of yeasts associated with corals and zoanthids in the Gulf of Thailand. Thirty-three coral and seven zoanthid samples were collected at two sampling sites near Mu and Khram islands. Fifty yeast strains were able to be isolated from 25 of the 40 samples collected. Identification based on sequence analyses of the D1/D2 domain of the large subunit rRNA gene revealed a higher number of strains in the phylum Basidiomycota (68%) than in the phylum Ascomycota. The ascomycetous yeasts comprised nine known species from four genera (Candida, Meyerozyma, Kodamaea, and Wickerhamomyces), whereas the basidiomycetous yeasts comprised 10 known species from eight genera (Vishniacozyma, Filobasidium, Naganishia, Papiliotrema, Sterigmatomyces, Cystobasidium, Rhodotorula, and Rhodosporidiobolus) and one potentially new species. The species with the highest occurrence was Rhodotorula mucilaginosa. Using principal coordinate analysis (PCoA) ordination, no marked differences were found in the yeast communities from the two sampling sites. The estimation of the expected richness of species was higher than the actual richness of species observed.

Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda : Parastacidae) inferred from four mitochondrial gene regions

2005 ◽  
Vol 19 (3) ◽  
pp. 209 ◽  
Author(s):  
Thuy T. T. Nguyen ◽  
Christopher M. Austin
Keyword(s):  
16S Rrna ◽  
Cytochrome Oxidase ◽  
Mitochondrial Gene ◽  
Large Subunit ◽  
Strong Support ◽  
Interspecific Variation ◽  
Rrna Gene ◽  
Freshwater Crayfish ◽  
Cherax Destructor ◽  
Australian Freshwater

The phylogenetic relationships among 32 individuals of Australian freshwater crayfish belonging to the Cherax destructor-complex were investigated using a dataset comprising sequences from four mitochondrial gene regions: the large subunit rRNA (16S rRNA), cytochrome oxidase I (COI), adenosine triphosphatase 6 (ATPase 6), and cytochrome oxidase III (COIII). A total of 1602 bp was obtained, and a combined analysis of the data produced a tree with strong support (bootstrap values 94–100%) for three divergent lineages, verifying the phylogenetic hypotheses of relationships within the C. destructor species-complex suggested in previous studies. Overall, sequences from the 16S rRNA gene showed the least variation compared to those generated from protein coding genes, which presented considerably greater levels of divergence. The level of divergence within C. destructor was found to be greater than that observed in other species of freshwater crayfish, but interspecific variation among species examined in the present study was similar to that reported previously.

scholarly journals Sympodiomyces attinorum sp. nov., a yeast species associated with nests of the leaf-cutting ant Atta sexdens

2004 ◽  
Vol 54 (5) ◽  
pp. 1891-1894 ◽  
Author(s):  
Solange C. Carreiro ◽  
Fernando C. Pagnocca ◽  
Maurício Bacci ◽  
Marc-André Lachance ◽  
Odair C. Bueno ◽  
...  
Keyword(s):  
Yeast Species ◽  
Novel Species ◽  
Large Subunit ◽  
Growth Characteristics ◽  
Rrna Gene ◽  
The Novel ◽  
Atta Sexdens ◽  
Leaf Cutting ◽  
Large Subunit Rrna ◽  
Waste Deposit

Four strains of a novel yeast species were isolated from laboratory nests of the leaf-cutting ant Atta sexdens in Brazil. Three strains were found in older sponges and one was in a waste deposit in the ant nests. Sequencing of the D1/D2 region of the large-subunit rRNA gene showed that the novel species, named Sympodiomyces attinorum sp. nov., is phylogenetically related to Sympodiomyces parvus. Unlike Sympodiomyces parvus, Sympodiomyces attinorum can ferment glucose, assimilate methyl α-d-glucoside, salicin and citrate, and grow at 37 °C, thus enabling these two species to be distinguished. Differentiation from other related species is possible on the basis of other growth characteristics. The type strain of Sympodiomyces attinorum is UNESP-S156T (=CBS 9734T=NRRL Y-27639T).

scholarly journals Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

2003 ◽  
Vol 69 (9) ◽  
pp. 5512-5518 ◽  
Author(s):  
Brett J. Baker ◽  
Philip Hugenholtz ◽  
Scott C. Dawson ◽  
Jillian F. Banfield
Keyword(s):  
Acid Mine Drainage ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Mine Drainage ◽  
Close Association ◽  
Variable Region ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Acid Mine

ABSTRACT During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “Candidatus Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

scholarly journals Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2080-2091 ◽  
Author(s):  
Anne-Laure Michon ◽  
Fabien Aujoulat ◽  
Laurent Roudière ◽  
Olivier Soulier ◽  
Isabelle Zorgniotti ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Diversity ◽  
Natural Populations ◽  
Variable Region ◽  
Housekeeping Gene ◽  
Single Copy ◽  
Population Based ◽  
Rrna Gene ◽  
Gene Copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

scholarly journals Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

scholarly journals Detection of Pneumocystis carinii DNA in Blood Specimens from Human Immunodeficiency Virus-Infected Patients by Nested PCR

1999 ◽  
Vol 37 (1) ◽  
pp. 127-131 ◽  
Author(s):  
Meja Rabodonirina ◽  
Laurent Cotte ◽  
André Boibieux ◽  
Karine Kaiser ◽  
Martine Mayençon ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Whole Blood ◽  
Nested Pcr ◽  
Large Subunit ◽  
Pneumocystis Carinii ◽  
Proteinase K ◽  
Rrna Gene ◽  
Immunodeficiency Virus ◽  
Blood Specimens ◽  
Large Subunit Rrna

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp.hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

scholarly journals Molecular characterisation of Leptospira strains in Pakistan

2016 ◽  
Vol 60 (4) ◽  
pp. 417-421
Author(s):  
Muhammad Luqman Sohail ◽  
Muhammad Sarwar Khan ◽  
Muhammad Avais ◽  
Muhammad Yasir Zahoor ◽  
Irfan Khattak ◽  
...  
Keyword(s):  
16S Rrna ◽  
Animal Species ◽  
Molecular Detection ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Intermediate Species ◽  
Specific Primers ◽  
Pathogenic Leptospira ◽  
Wide Range ◽  
Serological Studies

Abstract Introduction: Leptospirosis affects a wide range of mammals, humans, and even a few poikilothermic animal species. In Pakistan, serological studies of equine leptospirosis have reported a prevalence of over 40%, but no study has ever been conducted towards molecular detection of Leptospira in horses. Material and Methods: Blood samples from 128 horses were screened using ELISA and 41 positive samples were examined for the presence of leptospiral DNA using specific primers for 16S rRNA gene. Results: Out of 41 tested samples, 20 samples were found to be PCR-positive, revealing a fragment of 306 bp after gel electrophoresis. Sequencing and phylogenetic analysis of positive samples revealed circulation of pathogenic Leptospira spp. in Pakistani horses. No evidence of circulation of intermediate species was found in this study. Conclusion: This study reports the first molecular evidence of equine leptospirosis in Pakistan and lays ground for further research in this area. It also confirms the efficiency of 16S rRNA for the diagnosis of equine leptospirosis.

Usitatibacter rugosus gen. nov., sp. nov. and Usitatibacter palustris sp. nov., novel members of Usitatibacteraceae fam. nov. within the order Nitrosomonadales isolated from soil

2021 ◽  
Author(s):  
Selma Vieira ◽  
Katharina J. Huber ◽  
Meina Neumann-Schaal ◽  
Alicia Geppert ◽  
Manja Luckner ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Type Species ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
The Novel ◽  
Content Type ◽  
Link Type ◽  
Wide Range

Members of the metabolically diverse order Nitrosomonadales inhabit a wide range of environments. Two strains affiliated with this order were isolated from soils in Germany and characterized by a polyphasic approach. Cells of strains 0125_3T and Swamp67T are Gram-negative rods, non-motile, non-spore-forming, non-capsulated and divide by binary fission. They tested catalase-negative, but positive for cytochrome c-oxidase. Both strains form small white colonies on agar plates and grow aerobically and chemoorganotrophically on SSE/HD 1 : 10 medium, preferably utilizing organic acids and proteinaceous substrates. Strains 0125_3T and Swamp67T are mesophilic and grow optimally without NaCl addition at slightly alkaline conditions. Major fatty acids are C16 : 1  ω7c, C16 : 0 and C14 : 0. The major polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyglycerol. The predominant respiratory quinone is Q-8. The G+C content for 0125_3T and Swamp67T was 67 and 66.1 %, respectively. The 16S rRNA gene analysis indicated that the closest relatives (<91 % sequence similarity) of strain 0125_3T were Nitrosospira multiformis ATCC 25196T, Methyloversatilis universalis FAM5T and Denitratisoma oestradiolicum AcBE2-1T, while Nitrosospira multiformis ATCC 25196T, Nitrosospira tenuis Nv1T and Nitrosospira lacus APG3T were closest to strain Swamp67T. The two novel strains shared 97.4 % 16S rRNA gene sequence similarity with one another and show low average nucleotide identity of their genomes (83.8 %). Based on the phenotypic, chemotaxonomic, genomic and phylogenetic analysis, we propose the two novel species Usitatibacter rugosus sp. nov (type strain 0125_3T=DSM 104443T=LMG 29998T=CECT 9241T) and Usitatibacter palustris sp. nov. (type strain Swamp67T=DSM 104440T=LMG 29997T=CECT 9242T) of the novel genus Usitatibacter gen. nov., within the novel family Usitatibacteraceae fam. nov.

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence

1994 ◽  
Vol 14 (6) ◽  
pp. 4203-4215
Author(s):  
R Sweeney ◽  
L Chen ◽  
M C Yao
Keyword(s):  
Large Subunit ◽  
Sequence Divergence ◽  
Variable Region ◽  
Rrna Genes ◽  
Functional Importance ◽  
Functional Feature ◽  
Tertiary Structures ◽  
Unrelated Sequence ◽  
Evolutionarily Conserved ◽  
Large Subunit Rrna

Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.

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