Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Parasitology ◽  
2002 ◽  
Vol 124 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. B. A. MENDONÇA ◽  
N. S. NEHME ◽  
S. S. SANTOS ◽  
E. CUPOLILLO ◽  
N. VARGAS ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Ribosomal Rna ◽  
Southern Blotting ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pcr Products ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Ribosomal Cistron ◽  
Panstrongylus Geniculatus

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3–Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Sα rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5·8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Sα rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

scholarly journals Differentiation of ‘Candidatus Phytoplasma cynodontis’ Based on 16S rRNA and groEL Genes and Identification of a New Subgroup, 16SrXIV-C

Plant Disease ◽  
2015 ◽  
Vol 99 (11) ◽  
pp. 1578-1583 ◽  
Author(s):  
J. Mitrović ◽  
M. Smiljković ◽  
Erich Seemüller ◽  
Richard Reinhardt ◽  
Bruno Hüttel ◽  
...  
Keyword(s):  
16S Rrna ◽  
Ribosomal Rna ◽  
Phylogenetic Analyses ◽  
Rrna Gene ◽  
Groel Gene ◽  
Gene Sequence Analysis ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Insight Into

‘Candidatus Phytoplasma cynodontis’ is widespread in bermudagrass and has only been found in monocotyledonous plants. Molecular studies carried out on strains collected in Italy, Serbia, and Albania enabled verification of molecular variability in the 16S ribosomal RNA (rRNA) gene. Based on restriction fragment length polymorphism and sequence analyses, the strains from Serbia were clearly differentiated from all others and assigned to a new ribosomal DNA (rDNA) subgroup designated as 16SrXIV-C. A system for amplification of fragments containing the ‘Ca. P. cynodontis’ groEL gene was developed to enable study of its variability in related strains belonging to different 16SrXIV subgroups. Despite the fact that the groEL gene exhibited a greater sequence variation than 16S rRNA, the phylogenetic tree based on groEL gene sequence analysis was highly congruent with the 16S rDNA-based tree. The groEL gene analyses supported differentiation of the Serbian strains and definition of the new subgroup 16SrXIV-C. Phylogenetic analyses of both genes confirmed distinct phylogenetic lineages for strains belonging to 16SrXIV subgroups. Furthermore, groEL is the only nonribosomal marker developed for characterization of ‘Ca. P. cynodontis’ thus far, and its application in molecular surveys should provide better insight into the relationships among these phytoplasmas and correlation between strain differentiation and their geographical distribution.

scholarly journals Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil

2002 ◽  
Vol 68 (8) ◽  
pp. 3818-3829 ◽  
Author(s):  
Christopher Rösch ◽  
Alexander Mergel ◽  
Hermann Bothe
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nitrite Reductase ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Cloning And Sequencing ◽  
Genetic Traits ◽  
Uncultured Bacteria ◽  
Pcr Products ◽  
The 16S Rrna Gene

ABSTRACT Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd 1-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.

Characterization of the Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes: Monogenea) ribosomal intergenic spacer (IGS) DNA

Parasitology ◽  
2000 ◽  
Vol 121 (5) ◽  
pp. 555-563 ◽  
Author(s):  
C. M. COLLINS ◽  
C. O. CUNNINGHAM
Keyword(s):  
Ribosomal Rna ◽  
Tandem Repeats ◽  
Gene Array ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Sequence Motifs ◽  
Ribosomal Rna Gene ◽  
Intergenic Spacers ◽  
Transcription Start Sites ◽  
Pcr Products

The intergenic spacer of the ribosomal RNA gene array from the monogenean Gyrodactylus salaris was isolated using PCR amplification. PCR products were cloned and sequenced. Three different fragments of 0·63, 1·0 and 2·62 kb, were consistently obtained. These showed homology at the 5′ and 3′ termini but differed in their overall size and intervening sequence. The 5′ end showed homology to various 28S ribosomal RNA gene sequences, suggesting that this represented the 3′ terminus of the G. salaris 28S ribosomal RNA gene. A number of features common to other eukaryotic intergenic spacers were found in the longest sequence, including A + T rich sequences, palindromic sequences and tandemly repeated elements. Two regions of 23 bp sequences arranged in non-identical tandem repeats were identified. There were 9 repeats in both regions, separated by 81 bp of non-repetitive sequence. The repeat units from the two regions shared some similarity at their 3′ ends. The G. salaris intergenic spacer sequence was examined for sequence motifs involved in the transcription of the ribosomal RNA genes in other species. Several regions with homology to transcription start sites were identified.

scholarly journals Measuring and mitigating PCR bias in microbiota datasets

2021 ◽  
Vol 17 (7) ◽  
pp. e1009113
Author(s):  
Justin D. Silverman ◽  
Rachael J. Bloom ◽  
Sharon Jiang ◽  
Heather K. Durand ◽  
Eric Dallow ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Experimental Approach ◽  
Linear Models ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Human Gut ◽  
Pcr Bias ◽  
Integral Role ◽  
High Throughput Dna Sequencing ◽  
Log Ratio

PCR amplification plays an integral role in the measurement of mixed microbial communities via high-throughput DNA sequencing of the 16S ribosomal RNA (rRNA) gene. Yet PCR is also known to introduce multiple forms of bias in 16S rRNA studies. Here we present a paired modeling and experimental approach to characterize and mitigate PCR NPM-bias (PCR bias from non-primer-mismatch sources) in microbiota surveys. We use experimental data from mock bacterial communities to validate our approach and human gut microbiota samples to characterize PCR NPM-bias under real-world conditions. Our results suggest that PCR NPM-bias can skew estimates of microbial relative abundances by a factor of 4 or more, but that this bias can be mitigated using log-ratio linear models.

scholarly journals Persistent infections in chronic Chagas' disease patients treated with anti-Trypanosoma cruzi nitroderivatives

2000 ◽  
Vol 42 (3) ◽  
pp. 157-161 ◽  
Author(s):  
M. Socorro BRAGA ◽  
Liana LAURIA-PIRES ◽  
Enrique R. ARGAÑARAZ ◽  
Rubens J. NASCIMENTO ◽  
Antonio R. L. TEIXEIRA
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Study Groups ◽  
Persistent Infections ◽  
The Difference ◽  
Definition Of ◽  
Dna Primers ◽  
Chronic Chagas Disease

We used a molecular method and demonstrated that treatment of the chronic human Trypanosoma cruzi infections with nitroderivatives did not lead to parasitological cure. Seventeen treated and 17 untreated chronic Chagas' disease patients, with at least two out of three positive serologic assays for the infection, and 17 control subjects formed the study groups. PCR assays with nested sets of T. cruzi DNA primers monitored the efficacy of treatment. The amplification products were hybridized to their complementary internal sequences. Untreated and treated Chagas' disease patients yielded PCR amplification products with T. cruzi nuclear DNA primers. Competitive PCR was conducted to determine the quantity of parasites in the blood and revealed < 1 to 75 T. cruzi/ml in untreated (means 25.83 ± 26.32) and < 1 to 36 T. cruzi/ml in treated (means 6.45 ± 9.28) Chagas' disease patients. The difference between the means was not statistically significant. These findings reveal a need for precise definition of the role of treatment of chronic Chagas' disease patients with nitrofuran and nitroimidazole compounds.

scholarly journals Host-adaptation of the rare Enterocytozoon bieneusi genotype CHN4 in Myocastor coypus (Rodentia: Echimyidae) in China

2020 ◽  
Author(s):  
Fuchang Yu ◽  
Yangwenna Cao ◽  
Haiyan Wang ◽  
Qiang Liu ◽  
Aiyun Zhao ◽  
...  
Keyword(s):  
Infection Rate ◽  
Ribosomal Rna ◽  
Pcr Amplification ◽  
Its Region ◽  
Host Adaptation ◽  
Enterocytozoon Bieneusi ◽  
Rrna Gene ◽  
Myocastor Coypus ◽  
Gastrointestinal Pathogen ◽  
Infection Source

Abstract Background: Enterocytozoon bieneusi is a zoonotic gastrointestinal pathogen and can infect both humans and animals. The coypu (Myocastor coypus) is a semi-aquatic rodent, in which few E. bieneusi infections have been reported and the distribution of genotypes and zoonotic potential remains unknown.Methods: A total of 308 fresh fecal samples were collected from seven coypu farms in China to determine the infection rate and the distribution of genotypes of E. bieneusi from coypus using nested-PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene.Results: Enterocytozoon bieneusi was detected with an infection rate of 41.2% (n = 127). Four genotypes were identified, including three known genotypes (CHN4 (n = 111), EbpC (n = 8) and EbpA (n = 7)) and a novel genotype named CNCP1 (n = 1). Conclusions: The rare genotype CHN4 was the most common genotype in the present study, and the transmission dynamics of E. bieneusi in coypus were different from other rodents. To the best of our knowledge, this is the first report of E. bieneusi infections in coypus in China. Our study reveals that E. bieneusi in coypus may be a potential infection source to humans.

scholarly journals Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA

Plant Disease ◽  
1997 ◽  
Vol 81 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Seiichi Okuda ◽  
James P. Prince ◽  
Robert E. Davis ◽  
Ellen L. Dally ◽  
Ing-Ming Lee ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Gene Group ◽  
Beet Leafhopper ◽  
Pcr Products ◽  
Elm Yellows

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.

scholarly journals Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

2005 ◽  
Vol 71 (9) ◽  
pp. 5494-5500 ◽  
Author(s):  
Susan M. Barns ◽  
Christy C. Grow ◽  
Richard T. Okinaka ◽  
Paul Keim ◽  
Cheryl R. Kuske
Keyword(s):  
Related Species ◽  
Environmental Samples ◽  
Pcr Amplification ◽  
Soil Samples ◽  
Rrna Gene ◽  
New Subspecies ◽  
Pcr Products ◽  
Primer Sets ◽  
Initial Survey ◽  
Close Relatives

ABSTRACT Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

Presence of Meloidogyne enterolobii Yang & Eisenback (=M. mayaguensis) in guava and acerola from Costa Rica

Nematology ◽  
2012 ◽  
Vol 14 (2) ◽  
pp. 199-207 ◽  
Author(s):  
Danny Antonio Humphreys ◽  
Valerie Moroz Williamson ◽  
Luis Salazar ◽  
Lorena Flores-Chaves ◽  
Luis Gómez-Alpizar
Keyword(s):  
Mitochondrial Dna ◽  
Costa Rica ◽  
Ribosomal Rna ◽  
Pcr Amplification ◽  
Tropical Species ◽  
Repeat Region ◽  
Meloidogyne Enterolobii ◽  
Lateral Lines ◽  
Pcr Products ◽  
Single Fragment

The presence of Meloidogyne enterolobii on two hosts and in several locations in Costa Rica is reported. Meloidogyne spp. females were extracted from root of acerola and of wild and introduced guava and their perineal patterns were examined. Females from acerola showed round or dorso-ventrally ovoid perineal patterns, in some cases with lateral lines or with a moderately high to high dorsal arch as is characteristic of M. incognita. In guava, perineal patterns were similar, but with a low to moderate dorsal arch. PCR amplification of the mitochondrial DNA region between COII and the 16S ribosomal RNA yielded a unique product of 705 bp for the five populations extracted from both crops. In addition, the amplified product spanning the 63 bp repeat region of the mitochondrial genome was a single fragment of 320 bp in contrast to other tropical species, which typically amplify multiple bands. Size of PCR products of both mitochondrial regions and the sequence of the 63 bp repeat region supported the identity of these isolates as M. enterolobii (=M. mayaguensis).

scholarly journals Molecular Analysis of Carbon Monoxide-Oxidizing Bacteria Associated with Recent Hawaiian Volcanic Deposits

2004 ◽  
Vol 70 (7) ◽  
pp. 4242-4248 ◽  
Author(s):  
Kari E. Dunfield ◽  
Gary M. King
Keyword(s):  
Carbon Monoxide ◽  
Lava Flow ◽  
Large Subunit ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Clone Libraries ◽  
Tephra Deposit ◽  
Volcanic Deposits ◽  
Pcr Products ◽  
Phylogenetic Lineages

ABSTRACT Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase. The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest. PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans. PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ. On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct. Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers. On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations. Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration. Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity.

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