Screening of B chromosomes for presence of two genes in yellow-necked mice, Apodemus flavicollis (Mammalia, Rodentia)

B chromosomes (Bs) are a very heterogeneous group of extra chromosomes. In various species Bs occur with different nucleotide sequences ranging from repetitive to protein coding. In yellow-necked field mice, Apodemus flavicollis Bs are small euchromatic chromosomes and untill now, only few molecular analyses have been conducted. In this study we examined A. flavicollis individuals with different number of Bs for presence of two genes, C-KIT and 18S rRNA. The C-KIT proto-oncogene was found on Bs in three Canidae species and one Cervidae species. This gene is a coding receptor critical for proliferation and cell differentiation of hematopoietic, melanoblast and primordial germ cells, and is highly conserved within mammals. While using semiquantitative PCR, we did not notice any difference in the C-KIT band intensity among animals with different number of Bs (0-3). The presence of only one copy of C- KIT gene was confirmed using real time-PCR on genomic DNA of A. flavicollis specimens with different number of Bs. rRNA genes in eukaryotes? genome are organized like units of tandem repeated sequences. The units form distinct clusters on one to several chromosome pairs. rRNA genes were found on Bs in different species including two species of genus Apodemus. One particular sample with 2 Bs showed the number of 18S rRNA gene about three times that of the calibrator 0 B sample. This result can indicate the presence of 18S rRNA gene on Bs, but its confirmation requires the implementation of other methods. Still, we can neither confirm nor deny the existence of pseudogen of tested target genes, or lose of exon 1 of C-KIT protooncogen in Bs of A. flavicollis. Our findings are further discussed.

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Molecular Characterization of Ciliate Diversity in Stream Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.01438-07 ◽

2008 ◽

Vol 74 (6) ◽

pp. 1740-1747 ◽

Cited By ~ 52

Author(s):

Andrew Dopheide ◽

Gavin Lear ◽

Rebecca Stott ◽

Gillian Lewis

Keyword(s):

18S Rrna ◽

Pcr Primers ◽

18S Rrna Gene ◽

Sequence Diversity ◽

Rrna Genes ◽

Rrna Gene ◽

Sequencing Analysis ◽

Specific Pcr ◽

Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

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Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans

Applied and Environmental Microbiology ◽

10.1128/aem.02131-07 ◽

2008 ◽

Vol 74 (14) ◽

pp. 4336-4345 ◽

Cited By ~ 17

Author(s):

Christofer Troedsson ◽

Richard F. Lee ◽

Vivica Stokes ◽

Tina L. Walters ◽

Paolo Simonelli ◽

...

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Performance ◽

18S Rrna ◽

18S Rrna Gene ◽

Rrna Genes ◽

Rrna Gene ◽

Chromatography Method ◽

Factors Affecting ◽

Triethylammonium Acetate

ABSTRACT Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-paring technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63°C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.

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Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

Applied and Environmental Microbiology ◽

10.1128/aem.01644-14 ◽

2014 ◽

Vol 80 (17) ◽

pp. 5515-5521 ◽

Cited By ~ 22

Author(s):

Suzanne L. Ishaq ◽

André-Denis G. Wright

Keyword(s):

18S Rrna ◽

18S Rrna Gene ◽

Rrna Genes ◽

Rrna Gene ◽

Content Type ◽

Hypervariable Regions ◽

Blast Database ◽

Primer Sets ◽

18S Rrna Genes ◽

Ciliate Protozoa

ABSTRACTFour new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplifiedEntodinium simplexandOstracodiniumspp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the generaBandia,Blepharocorys,Polycosta, andTetratoxumand betweenHemiprorodon gymnoprosthiumandProrodonopsiscoli, none of which are normally found in the rumen.

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Molecular identification ofSarcocystisspp. helped to define the origin of green pythons (Morelia viridis) confiscated in Germany

Parasitology ◽

10.1017/s0031182013001960 ◽

2013 ◽

Vol 141 (5) ◽

pp. 646-651 ◽

Cited By ~ 9

Author(s):

GASTÓN MORÉ ◽

NIKOLA PANTCHEV ◽

DALAND C. HERRMANN ◽

MAJDA GLOBOKAR VRHOVEC ◽

SABINE ÖFNER ◽

...

Keyword(s):

18S Rrna ◽

Sequence Data ◽

18S Rrna Gene ◽

Rrna Genes ◽

Rrna Gene ◽

Intermediate Hosts ◽

Apicomplexan Parasites ◽

Large Numbers ◽

Wide Range ◽

18S Rrna Genes

SUMMARYSarcocystisspp. represent apicomplexan parasites. They usually have a heteroxenous life cycle. Around 200 species have been described, affecting a wide range of animals worldwide, including reptiles. In recent years, large numbers of reptiles have been imported into Europe as pets and, as a consequence, animal welfare and species protection issues emerged. A sample of pooled feces from four confiscated green pythons (Morelia viridis) containingSarcocystisspp. sporocysts was investigated. These snakes were imported for the pet trade and declared as being captive-bred. Full length 18S rRNA genes were amplified, cloned into plasmids and sequenced. Two differentSarcocystisspp. sequences were identified and registered asSarcocystissp. fromM. viridisin GenBank. Both showed a 95–97% sequence identity with the 18S rRNA gene ofSarcocystis singaporensis.Phylogenetic analysis positioned these sequences together with otherSarcocystisspp. from snakes and rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakes were definitive hosts forSarcocystisspp. that circulate in wild IH. Thus, it seems unlikely that the infected snakes had been legally bred. Our research shows that information on the infection of snakes withSarcocystisspp. may be used to assess compliance with regulations on the trade with wildlife species.

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Mitogenomics and the Phylogeny of Mantis Shrimps (Crustacea: Stomatopoda)

Diversity ◽

10.3390/d13120647 ◽

2021 ◽

Vol 13 (12) ◽

pp. 647

Author(s):

Cassandra Koga ◽

Greg W. Rouse

Keyword(s):

Sister Group ◽

18S Rrna Gene ◽

16S Rrna Genes ◽

Rrna Genes ◽

Mitochondrial Genomes ◽

12S Rrna ◽

Rrna Gene ◽

Slight Variation ◽

Ancestral State ◽

Protein Coding

Stomatopoda, commonly known as mantis shrimps, are notable for their enlarged second maxillipeds encompassing the raptorial claw. The form of the claw can be used to divide them into two basic groups: smashers and spearers. Previous phylogenetic studies of Stomatopoda have focused on morphology or a few genes, though there have been whole mitochondrial genomes published for 15 members of Stomatopoda. However, the sampling has been somewhat limited with key taxa not included. Here, nine additional stomatopod mitochondrial genomes were generated and combined with the other available mitogenomes for a phylogenetic analysis. We used the 13 protein coding genes, as well as 12S rRNA, 16S rRNA genes, and included nuclear 18S rRNA gene sequences. Different rooting options were used for the analyses: (1) single and multiple outgroups from various eumalocostracan relatives and (2) a stomatopod-only dataset, with Hemisquilla californiensis used to root the topologies, based on the current hypothesis that Hemisquilla is the sister group to the rest of Stomatopoda. The eumalocostracan-rooted analyses all showed H. californiensis nested within Stomatopoda, raising doubts as to previous hypotheses as to its placement. Allowing for the rooting difference, the H. californiensis outgroup datasets had the same tree topology as the eumalocostracan outgroup datasets with slight variation at poorly supported nodes. Of the major taxonomic groupings sampled to date, Squilloidea was generally found to be monophyletic while Gonodactyloidea was not. The position of H. californiensis was found inside its superfamily, Gonodactyloidea, and grouped in a weakly supported clade containing Odontodactylus havanensis and Lysiosquillina maculata for the eumalocostracan-rooted datasets. An ancestral state reconstruction was performed on the raptorial claw form and provides support that spearing is the ancestral state for extant Stomatopoda, with smashing evolving subsequently one or more times.

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Dogs are reservoir hosts for possible transmission of human strongyloidiasis in Thailand: molecular identification and genetic diversity of causative parasite species

Journal of Helminthology ◽

10.1017/s0022149x1900107x ◽

2019 ◽

Vol 94 ◽

Cited By ~ 3

Author(s):

O. Sanpool ◽

P.M. Intapan ◽

R. Rodpai ◽

P. Laoraksawong ◽

L. Sadaow ◽

...

Keyword(s):

18S Rrna ◽

Gastrointestinal Disease ◽

Close Association ◽

Lao Pdr ◽

Strongyloides Stercoralis ◽

18S Rrna Gene ◽

Rrna Genes ◽

Rrna Gene ◽

North East ◽

18S Rrna Genes

Abstract Human strongyloidiasis is a deleterious gastrointestinal disease mainly caused by Strongyloides stercoralis infection. We aimed to study the possible transmission of S. stercoralis between humans and pet animals. We isolated Strongyloides from humans and domestic dogs in the same rural community in north-east Thailand and compared the nucleotide sequences of derived worms using portions of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and 18S ribosomal RNA (18S rRNA) genes. Twenty-eight sequences from the 18S rRNA gene were obtained from worms derived from humans (n = 23) and dogs (n = 5), and were identical with S. stercoralis sequences (from Thailand, Cambodia, Lao PDR and Myanmar) published in the GenBank database. The 28 cox1 sequences from humans and dogs showed high similarity to each other. The available published cox1 sequences (n = 150), in combination with our 28 sequences, represented 68 haplotypes distributed among four clusters. The 28 samples from the present study represented eight haplotypes including four new haplotypes. Dogs and humans shared the same haplotypes, suggesting the possibility of zoonotic transmission from pet dogs to humans. This is of concern since dogs and humans live in close association with each other.

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Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

Applied and Environmental Microbiology ◽

10.1128/aem.01630-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5878-5891 ◽

Cited By ~ 66

Author(s):

Ian M. Bradley ◽

Ameet J. Pinto ◽

Jeremy S. Guest

Keyword(s):

Community Structure ◽

18S Rrna ◽

Target Genes ◽

Hypervariable Region ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

18S Rrna Gene ◽

Rrna Gene ◽

Sequencing Bias ◽

Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

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18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

Applied and Environmental Microbiology ◽

10.1128/aem.69.9.5389-5397.2003 ◽

2003 ◽

Vol 69 (9) ◽

pp. 5389-5397 ◽

Cited By ~ 62

Author(s):

Zhihong Wu ◽

Yoshihiko Tsumura ◽

Göran Blomquist ◽

Xiao-Ru Wang

Keyword(s):

18S Rrna ◽

Airborne Fungi ◽

18S Rrna Gene ◽

Rrna Genes ◽

Molecular Diagnostic ◽

Rrna Gene ◽

Oligonucleotide Probes ◽

Gene Variation ◽

Species Specific ◽

18S Rrna Genes

ABSTRACT In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.

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The B chromosomes of the African cichlid fish Haplochromis obliquidens harbour 18S rRNA gene copies

BMC Genetics ◽

10.1186/1471-2156-11-1 ◽

2010 ◽

Vol 11 (1) ◽

pp. 1 ◽

Cited By ~ 56

Author(s):

Andréia B Poletto ◽

Irani A Ferreira ◽

Cesar Martins

Keyword(s):

18S Rrna ◽

Cichlid Fish ◽

18S Rrna Gene ◽

B Chromosomes ◽

Rrna Gene ◽

African Cichlid ◽

Gene Copies

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Babesia microti in Rodents from Different Habitats of Lithuania

Animals ◽

10.3390/ani11061707 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1707

Author(s):

Dalytė Mardosaitė-Busaitienė ◽

Jana Radzijevskaja ◽

Linas Balčiauskas ◽

Algimantas Paulauskas

Keyword(s):

18S Rrna ◽

18S Rrna Gene ◽

Reservoir Host ◽

Clethrionomys Glareolus ◽

Babesia Microti ◽

Rrna Gene ◽

Apodemus Flavicollis ◽

Study Sites ◽

First Time ◽

Partial 18S

Babesia microti (Aconoidasida: Piroplasmida) (Franca, 1910) is an emerging tick-borne parasite with rodents serving as the considered reservoir host. However, the distribution of B. microti in Europe is insufficiently characterized. Based on the sample of 1180 rodents from 19 study sites in Lithuania, the objectives of this study were: (1) to investigate the presence of Babesia parasites in eight species of rodents, (2) to determine the prevalence of Babesia parasites in rodents from different habitats, and (3) to characterize the detected Babesia strains using partial sequencing of the 18S rRNR gene. Babesia DNA was detected in 2.8% rodents. The highest prevalence of Babesia was found in Microtus oeconomus (14.5%) and Microtus agrestis (7.1%) followed by Clethrionomys glareolus (2.3%), Apodemus flavicollis (2.2%) and Micromys minutus (1.3%). In M. minutus, Babesia was identified for the first time. The prevalence of Babesia-infected rodents was higher in the meadow (5.67%) than in the ecotone (1.69%) and forest (0.31%) habitats. The sequence analysis of the partial 18S rRNA gene reveals that Babesia isolates derived from rodents were 99–100% identical to human pathogenic B. microti ‘Jena/Germany’ strain.

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