Eukaryotic diversity in premise drinking water using 18S rDNA sequencing: implications for health risks

EPA Science Inventory

The goal of this study was to characterize microbial eukaryotes over a 12 month period, so as to provide insight into the occurrence of potentially important predators and bacterial hosts in hot and cold premise plumbing. Nearly 6,300 partial (600 bp) 18S rRNA gene sequences from...

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PubMed

Straub, Shannon C K; Parks, Matthew; Weitemier, Kevin; Fishbein, Mark; Cronn, Richard C; Liston, Aaron

2012-02-01

Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Morphological and molecular genetic diversity of Strongyluris calotis (Nematoda: Ascaridida: Heterakidae) in South East and East Asian lizards.

PubMed

Tran, Binh Thi; Ong, An Vinh; Luc, Pham Van; Sato, Hiroshi

2016-07-01

Strongyluris calotis is a heterakid nematode in the large intestine of agamid lizards (Reptilia: Sauria: Agamidae) from the Oriental Region. The standard light microscopic definition of the species counts the "caudal papillae" as 10 pairs on male worms. However, previous work from our group using scanning electron microscopy (SEM) on the heterakid from agamid lizards in Japan, Taiwan, and Singapore revealed that this counting contained a pair of phasmids and that two pairs of postcloacal papillae were completely fused to form a pair of united papillae, thus resulting in "10 pairs." In the present study, we examined S. calotis specimens from the Emma Gray's forest lizard, Calotes emma (Agamidae), living in the plain forest at low altitude, and the Vietnam false bloodsucker, Pseudocalotes brevipes (Agamidae), living in the mountainous forest at high altitude in the northern part of Vietnam. Using SEM, the arrangement of caudal papillae in male worms from an Emma Gray's forest lizard was found to be comparable to classical S. calotis specimens from agamid lizards collected in Japan, Taiwan, and Singapore. However, male worms from Vietnam false bloodsuckers did not have a pair of united papillae but had 10 pairs of independent caudal papillae with a pair of phasmids. Molecular genetic analyses of the ribosomal RNA gene (rDNA) of worms of the classical S. calotis morphotype from Japan and Singapore and two S. calotis morphotypes from Vietnam demonstrated absolutely identical nucleotide sequences of partial 18S rDNA (at least 1764 base pairs (bp)) and 5.8S rDNA (158 bp). However, intraspecific differences were detected in other regions of the rDNA, related to the geographical distribution of hosts regardless of morphotype: 97.8-98.5 % identity (443-446 bp/453 bp) in the internal transcribed spacer (ITS)-1 region, 96.6-98.0 % identity (425-431 bp/440 bp) in the ITS-2 region, and 99.6-99.7 % identity (1149-1151 bp/1154 bp) in the 28S rDNA. Thus, in the future, taxonomic relationships of S. calotis distributed widely in the Oriental Region as well as other nominal Oriental Strongyluris spp., currently six in number, need to be extensively explored based on molecular genetic analyses in addition to intensive morphological characterization.

Phylogenetic position of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, as inferred from 16S rDNA sequence analysis.

PubMed

Atibalentja, N; Noel, G R; Domier, L L

2000-03-01

A 1341 bp sequence of the 16S rDNA of an undescribed species of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, was determined and then compared with a homologous sequence of Pasteuria ramosa, a parasite of cladoceran water fleas of the family Daphnidae. The two Pasteuria sequences, which diverged from each other by a dissimilarity index of 7%, also were compared with the 16S rDNA sequences of 30 other bacterial species to determine the phylogenetic position of the genus Pasteuria among the Gram-positive eubacteria. Phylogenetic analyses using maximum-likelihood, maximum-parsimony and neighbour-joining methods showed that the Heterodera glycines-infecting Pasteuria and its sister species, P. ramosa, form a distinct line of descent within the Alicyclobacillus group of the Bacillaceae. These results are consistent with the view that the genus Pasteuria is a deeply rooted member of the Clostridium-Bacillus-Streptococcus branch of the Gram-positive eubacteria, neither related to the actinomycetes nor closely related to true endospore-forming bacteria.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

PubMed

Chelomina, Galina N; Rozhkovan, Konstantin V; Voronova, Anastasia N; Burundukova, Olga L; Muzarok, Tamara I; Zhuravlev, Yuri N

2016-04-01

Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

PubMed Central

Chelomina, Galina N.; Rozhkovan, Konstantin V.; Voronova, Anastasia N.; Burundukova, Olga L.; Muzarok, Tamara I.; Zhuravlev, Yuri N.

2015-01-01

Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine. PMID:27158239

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

PubMed

Lakshmikumaran, M; Negi, M S

1994-03-01

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

In and out of the rRNA genes: characterization of Pokey elements in the sequenced Daphnia genome

PubMed Central

2013-01-01

Background Only a few transposable elements are known to exhibit site-specific insertion patterns, including the well-studied R-element retrotransposons that insert into specific sites within the multigene rDNA. The only known rDNA-specific DNA transposon, Pokey (superfamily: piggyBac) is found in the freshwater microcrustacean, Daphnia pulex. Here, we present a genome-wide analysis of Pokey based on the recently completed whole genome sequencing project for D. pulex. Results Phylogenetic analysis of Pokey elements recovered from the genome sequence revealed the presence of four lineages corresponding to two divergent autonomous families and two related lineages of non-autonomous miniature inverted repeat transposable elements (MITEs). The MITEs are also found at the same 28S rRNA gene insertion site as the Pokey elements, and appear to have arisen as deletion derivatives of autonomous elements. Several copies of the full-length Pokey elements may be capable of producing an active transposase. Surprisingly, both families of Pokey possess a series of 200 bp repeats upstream of the transposase that is derived from the rDNA intergenic spacer (IGS). The IGS sequences within the Pokey elements appear to be evolving in concert with the rDNA units. Finally, analysis of the insertion sites of Pokey elements outside of rDNA showed a target preference for sites similar to the specific sequence that is targeted within rDNA. Conclusions Based on the target site preference of Pokey elements and the concerted evolution of a segment of the element with the rDNA unit, we propose an evolutionary path by which the ancestors of Pokey elements have invaded the rDNA niche. We discuss how specificity for the rDNA unit may have evolved and how this specificity has played a role in the long-term survival of these elements in the subgenus Daphnia. PMID:24059783

[Investigation of bacterial diversity in the biological desulfurization reactor for treating high salinity wastewater by the 16S rDNA cloning method].

PubMed

Liu, Wei-Guo; Liang, Cun-Zhen; Yang, Jin-Sheng; Wang, Gui-Ping; Liu, Miao-Miao

2013-02-01

The bacterial diversity in the biological desulfurization reactor operated continuously for 1 year was studied by the 16S rDNA cloning and sequencing method. Forty clones were randomly selected and their partial 16S rDNA genes (ca. 1,400 bp) were sequenced and blasted. The results indicated that there were dominant bacterias in the biological desulfurization reactor, where 33 clones belonged to 3 different published phyla, while 1 clone belonged to unknown phylum. The dominant bacterial community in the system was Proteobacteria, which accounted for 85.3%. The bacterial community succession was as follows: the gamma-Proteobacteria(55.9%), beta-Proteobacteria(17.6%), Actinobacteridae (8.8%), delta-Proteobacteria (5.9%) , alpha-Proteobacteria(5.9%), and Sphingobacteria (2.9%). Halothiobacillus sp. ST15 and Thiobacillus sp. UAM-I were the major desulfurization strains.

Phylogenetic relationships in three species of canine Demodex mite based on partial sequences of mitochondrial 16S rDNA.

PubMed

Sastre, Natalia; Ravera, Ivan; Villanueva, Sergio; Altet, Laura; Bardagí, Mar; Sánchez, Armand; Francino, Olga; Ferrer, Lluís

2012-12-01

The historical classification of Demodex mites has been based on their hosts and morphological features. Genome sequencing has proved to be a very effective taxonomic tool in phylogenetic studies and has been applied in the classification of Demodex. Mitochondrial 16S rDNA has been demonstrated to be an especially useful marker to establish phylogenetic relationships. To amplify and sequence a segment of the mitochondrial 16S rDNA from Demodex canis and Demodex injai, as well as from the short-bodied mite called, unofficially, D. cornei and to determine their genetic proximity. Demodex mites were examined microscopically and classified as Demodex folliculorum (one sample), D. canis (four samples), D. injai (two samples) or the short-bodied species D. cornei (three samples). DNA was extracted, and a 338 bp fragment of the 16S rDNA was amplified and sequenced. The sequences of the four D. canis mites were identical and shared 99.6 and 97.3% identity with two D. canis sequences available at GenBank. The sequences of the D. cornei isolates were identical and showed 97.8, 98.2 and 99.6% identity with the D. canis isolates. The sequences of the two D. injai isolates were also identical and showed 76.6% identity with the D. canis sequence. Demodex canis and D. injai are two different species, with a genetic distance of 23.3%. It would seem that the short-bodied Demodex mite D. cornei is a morphological variant of D. canis. © 2012 The Authors. Veterinary Dermatology © 2012 ESVD and ACVD.

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

PubMed

Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

2012-05-01

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. Copyright © 2012 Elsevier Inc. All rights reserved.

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

PubMed

Negi, Madan S; Rajagopal, Jyothi; Chauhan, Neeti; Cronn, Richard; Lakshmikumaran, Malathi

2002-12-01

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.

Molecular cloning and analysis of Schizosaccharomyces pombe Reb1p: sequence-specific recognition of two sites in the far upstream rDNA intergenic spacer.

PubMed Central

Zhao, A; Guo, A; Liu, Z; Pape, L

1997-01-01

The coding sequences for a Schizosaccharomyces pombe sequence-specific DNA binding protein, Reb1p, have been cloned. The predicted S. pombe Reb1p is 24-29% identical to mouse TTF-1 (transcription termination factor-1) and Saccharomyces cerevisiae REB1 protein, both of which direct termination of RNA polymerase I catalyzed transcripts. The S.pombe Reb1 cDNA encodes a predicted polypeptide of 504 amino acids with a predicted molecular weight of 58.4 kDa. The S. pombe Reb1p is unusual in that the bipartite DNA binding motif identified originally in S.cerevisiae and Klyveromyces lactis REB1 proteins is uninterrupted and thus S.pombe Reb1p may contain the smallest natural REB1 homologous DNA binding domain. Its genomic coding sequences were shown to be interrupted by two introns. A recombinant histidine-tagged Reb1 protein bearing the rDNA binding domain has two homologous, sequence-specific binding sites in the S. pomber DNA intergenic spacer, located between 289 and 480 nt downstream of the end of the approximately 25S rRNA coding sequences. Each binding site is 13-14 bp downstream of two of the three proposed in vivo termination sites. The core of this 17 bp site, AGGTAAGGGTAATGCAC, is specifically protected by Reb1p in footprinting analysis. PMID:9016645

Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish.

PubMed

Glugoski, Larissa; Giuliano-Caetano, Lucia; Moreira-Filho, Orlando; Vicari, Marcelo R; Nogaroto, Viviane

2018-04-15

Co-located 5S rDNA genes and interstitial telomeric sites (ITS) revealed the involvement of multiple 5S rDNA clusters in chromosome rearrangements of Loricariidae. Interstitial (TTAGGG)n vestiges, in addition to telomeric sites, can coincide with locations of chromosomal rearrangements, and they are considered to be hotspots for chromosome breaks. This study aimed the molecular characterization of 5S rDNA in two Rineloricaria latirostris populations and examination of roles of 5S rDNA in breakpoint sites and its in situ localization. Rineloricaria latirostris from Brazil's Das Pedras river (2n = 46 chromosomes) presented five pairs identified using a 5S rDNA probe, in addition to a pair bearing a co-located ITS/5S rDNA. Rineloricaria latirostris from the Piumhi river (2n = 48 chromosomes) revealed two pairs containing 5S rDNA, without ITS. A 702-bp amplified sequence, using 5S rDNA primers, revealed an insertion of the hAT transposable element (TE), referred to as a degenerate 5S rDNA. Double-FISH (fluorescence in situ hybridization) demonstrated co-localization of 5S rDNA/degenerate 5S rDNA, 5S rDNA/hAT and ITS/5S rDNA from the Das Pedras river population. Piumhi river isolates possessed only 5S rDNA sites. We suggest that the degenerate 5S rDNA was generated by unequal crossing over, which was driven by invasion of hAT, establishing a breakpoint region susceptible to chromosome breakage, non-homologous recombination and Robertsonian (Rb) fusion. Furthermore, the presence of clusters of 5S rDNA at fusion points in other armored catfish species suggests its re-use and that these regions represent hotspots for evolutionary rearrangements within Loricariidae genomes. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel variants of the 5S rRNA genes in Eruca sativa.

PubMed

Singh, K; Bhatia, S; Lakshmikumaran, M

1994-02-01

The 5S ribosomal RNA (rRNA) genes of Eruca sativa were cloned and characterized. They are organized into clusters of tandemly repeated units. Each repeat unit consists of a 119-bp coding region followed by a noncoding spacer region that separates it from the coding region of the next repeat unit. Our study reports novel gene variants of the 5S rRNA genes in plants. Two families of the 5S rDNA, the 0.5-kb size family and the 1-kb size family, coexist in the E. sativa genome. The 0.5-kb size family consists of the 5S rRNA genes (S4) that have coding regions similar to those of other reported plant 5S rDNA sequences, whereas the 1-kb size family consists of the 5S rRNA gene variants (S1) that exist as 1-kb BamHI tandem repeats. S1 is made up of two variant units (V1 and V2) of 5S rDNA where the BamHI site between the two units is mutated. Sequence heterogeneity among S4, V1, and V2 units exists throughout the sequence and is not limited to the noncoding spacer region only. The coding regions of V1 and V2 show approximately 20% dissimilarity to the coding regions of S4 and other reported plant 5S rDNA sequences. Such a large variation in the coding regions of the 5S rDNA units within the same plant species has been observed for the first time. Restriction site variation is observed between the two size classes of 5S rDNA in E. sativa.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular detection and characterization of Anaplasma platys in dogs and ticks in Cuba.

PubMed

Silva, Claudia Bezerra da; Santos, Huarrisson Azevedo; Navarrete, Maylín González; Ribeiro, Carla Carolina Dias Uzedo; Gonzalez, Belkis Corona; Zaldivar, Maykelin Fuentes; Pires, Marcus Sandes; Peckle, Maristela; Costa, Renata Lins da; Vitari, Gabriela Lopes Vivas; Massard, Carlos Luiz

2016-07-01

Canine cyclic thrombocytopenia, an infectious disease caused by Anaplasma platys is a worldwide dog health problem. This study aimed to detect and characterize A. platys deoxyribonucleic acid (DNA) in dogs and ticks from Cuba using molecular methods. The study was conducted in four cities of Cuba (Habana del Este, Boyeros, Cotorro and San José de las Lajas). Blood samples were collected from 100 dogs in these cities. The animals were inspected for the detection of tick infestation and specimens were collected. Genomic DNA was extracted from dog blood and ticks using a commercial kit. Genomic DNA samples from blood and ticks were tested by a nested polymerase chain reaction (nPCR) to amplify 678 base pairs (bp) from the 16S ribosomal DNA (rDNA) of A. platys. Positive samples in nPCR were also subjected to PCR to amplify a fragment of 580bp from the citrate synthase (gltA) gene and the products were sequenced. Only Rhipicephalus sanguineus sensu lato (s.l.) was found on dogs, and 10.20% (n=5/49) of these ticks plus sixteen percent (16.0%, n=16/100) of dogs were considered positive for A. platys by nPCR targeting the 16S rDNA gene. All analyzed gltA and 16S rDNA sequences showed a 99-100% identity with sequences of A. platys reported in around the world. Phylogenetic analysis showed two defined clusters for the 16S rDNA gene and three defined clusters for the gltA gene. Based on the gltA gene, the deduced amino acid sequence showed two mutations at positions 88 and 168 compared with the sequence DQ525687 (GenBank ID from Italian sample), used as a reference in the alignment. A preliminary study on the epidemiological aspects associated with infection by A. platys showed no statistical association with the variables studied (p>0.05). This is the first evidence of the presence of A. platys in dogs and ticks in Cuba. Further studies are needed to evaluate the epidemiological aspects of A. platys infection in Cuban dogs. Copyright © 2016 Elsevier GmbH. All rights reserved.

Studying long 16S rDNA sequences with ultrafast-metagenomic sequence classification using exact alignments (Kraken).

PubMed

Valenzuela-González, Fabiola; Martínez-Porchas, Marcel; Villalpando-Canchola, Enrique; Vargas-Albores, Francisco

2016-03-01

Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database.

PubMed

Cloud, Joann L; Conville, Patricia S; Croft, Ann; Harmsen, Dag; Witebsky, Frank G; Carroll, Karen C

2004-02-01

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5' 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i). conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii). molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii). when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences.

PubMed

Przyboś, Ewa; Tarcz, Sebastian; Greczek-Stachura, Magdalena; Surmacz, Marta

2011-05-01

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice-natural pond; Jordan's Park-artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies. Copyright © 2010 Elsevier GmbH. All rights reserved.

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

PubMed

Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A

2017-11-14

The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

The formation of diploid and triploid hybrids of female grass carp × male blunt snout bream and their 5S rDNA analysis.

PubMed

He, Weiguo; Xie, Lihua; Li, Tangluo; Liu, Shaojun; Xiao, Jun; Hu, Jie; Wang, Jing; Qin, Qinbo; Liu, Yun

2013-11-23

Hybridization is a useful strategy to alter the genotypes and phenotypes of the offspring. It could transfer the genome of one species to another through combing the different genome of parents in the hybrid offspring. And the offspring may exhibit advantages in growth rate, disease resistance, survival rate and appearance, which resulting from the combination of the beneficial traits from both parents. Diploid and triploid hybrids of female grass carp (Ctenopharyngodon idellus, GC, Cyprininae, 2n = 48) × male blunt snout bream (Megalobrama amblycephala, BSB, Cultrinae, 2n = 48) were successfully obtained by distant hybridization. Diploid hybrids had 48 chromosomes, with one set from GC and one set from BSB. Triploid hybrids possessed 72 chromosomes, with two sets from GC and one set from BSB.The morphological traits, growth rates, and feeding ecology of the parents and hybrid offspring were compared and analyzed. The two kinds of hybrid offspring exhibited significantly phenotypic divergence from GC and BSB. 2nGB hybrids showed similar growth rate compared to that of GC, and 3nGB hybrids significantly higher results. Furthermore, the feeding ecology of hybrid progeny was omnivorous.The 5S rDNA of GC, BSB and their hybrid offspring were also cloned and sequenced. There was only one type of 5S rDNA (designated type I: 180 bp) in GC and one type of 5S rDNA (designated type II: 188 bp) in BSB. However, in the hybrid progeny, diploid and triploid hybrids both inherited type I and type II from their parents, respectively. In addition, a chimera of type I and type II was observed in the genome of diploid and triploid hybrids, excepting a 10 bp of polyA insertion in type II sequence of the chimera of the diploid hybrids. This is the first report of diploid and triploid hybrids being produced by crossing GC and BSB, which have the same chromosome number. The obtainment of two new hybrid offspring has significance in fish genetic breeding. The results illustrate the effect of hybridization and polyploidization on the organization and variation of 5S rDNA in hybrid offspring.

In silico analysis of 16S ribosomal RNA gene sequencing‐based methods for identification of medically important anaerobic bacteria

PubMed Central

Woo, Patrick C Y; Chung, Liliane M W; Teng, Jade L L; Tse, Herman; Pang, Sherby S Y; Lau, Veronica Y T; Wong, Vanessa W K; Kam, Kwok‐ling; Lau, Susanna K P; Yuen, Kwok‐Yung

2007-01-01

This study is the first study that provides useful guidelines to clinical microbiologists and technicians on the usefulness of full 16S rRNA sequencing, 5′‐end 527‐bp 16S rRNA sequencing and the existing MicroSeq full and 500 16S rDNA bacterial identification system (MicroSeq, Perkin‐Elmer Applied Biosystems Division, Foster City, California, USA) databases for the identification of all existing medically important anaerobic bacteria. Full and 527‐bp 16S rRNA sequencing are able to identify 52–63% of 130 Gram‐positive anaerobic rods, 72–73% of 86 Gram‐negative anaerobic rods and 78% of 23 anaerobic cocci. The existing MicroSeq databases are able to identify only 19–25% of 130 Gram‐positive anaerobic rods, 38% of 86 Gram‐negative anaerobic rods and 39% of 23 anaerobic cocci. These represent only 45–46% of those that should be confidently identified by full and 527‐bp 16S rRNA sequencing. To improve the usefulness of MicroSeq, bacterial species that should be confidently identified by full and/or 527‐bp 16S rRNA sequencing but not included in the existing MicroSeq databases should be included. PMID:17046845

Nucleolar Association and Transcriptional Inhibition through 5S rDNA in Mammals

PubMed Central

Fedoriw, Andrew M.; Starmer, Joshua; Yee, Della; Magnuson, Terry

2012-01-01

Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals. PMID:22275877

Variations in 5S rDNAs in diploid and tetraploid offspring of red crucian carp × common carp.

PubMed

Ye, Lihai; Zhang, Chun; Tang, Xiaojun; Chen, Yiyi; Liu, Shaojun

2017-08-08

The allotetraploid hybrid fish (4nAT) that was created in a previous study through an intergeneric cross between red crucian carp (Carassius auratus red var., ♀) and common carp (Cyprinus carpio L., ♂) provided an excellent platform to investigate the effect of hybridization and polyploidization on the evolution of 5S rDNA. The 5S rDNAs of paternal common carp were made up of a coding sequence (CDS) and a non-transcribed spacer (NTS) unit, and while the 5S rDNAs of maternal red crucian carp contained a CDS and a NTS unit, they also contained a variable number of interposed regions (IPRs). The CDSs of the 5S rDNAs in both parental fishes were conserved, while their NTS units seemed to have been subjected to rapid evolution. The diploid hybrid 2nF 1 inherited all the types of 5S rDNAs in both progenitors and there were no signs of homeologous recombination in the 5S rDNAs of 2nF 1 by sequencing of PCR products. We obtained two segments of 5S rDNA with a total length of 16,457 bp from allotetraploid offspring 4nAT through bacterial artificial chromosome (BAC) sequencing. Using this sequence together with the 5S rDNA sequences amplified from the genomic DNA of 4nAT, we deduced that the 5S rDNAs of 4nAT might be inherited from the maternal progenitor red crucian carp. Additionally, the IPRs in the 5S rDNAs of 4nAT contained A-repeats and TA-repeats, which was not the case for the IPRs in the 5S rDNAs of 2nF 1 . We also detected two signals of a 200-bp fragment of 5S rDNA in the chromosomes of parental progenitors and hybrid progenies by fluorescence in situ hybridization (FISH). We deduced that during the evolution of 5S rDNAs in different ploidy hybrid fishes, interlocus gene conversion events and tandem repeat insertion events might occurred in the process of polyploidization. This study provided new insights into the relationship among the evolution of 5S rDNAs, hybridization and polyploidization, which were significant in clarifying the genome evolution of polyploid fish.

Incidence of three Kudoa spp., K. neothunni, K. hexapunctata, and K. thunni (Myxosporea: Multivalvulida), in Thunnus tunas distributed in the western Pacific Ocean.

PubMed

Kasai, Akihiro; Tsuduki, Hideaki; Jimenez, Lea Angsinco; Li, Ying-Chun; Tanaka, Shuhei; Sato, Hiroshi

2017-04-01

A variety of tunas of the genus Thunnus are consumed daily in Japan as sliced raw fish (sashimi and sushi). The consumption of fresh sliced raw fish, i.e., unfrozen or uncooked, can sometimes cause food poisoning that is manifested by transient diarrhea and vomiting for a single day. One of the causes of this type of food poisoning has been identified as live Kudoa septempunctata (Myxosporea: Multivalvulida) in the olive flounder (Paralichthys olivaceus). Furthermore, raw slices of fresh tunas are highly suspected to be a possible causative fish of similar food poisoning in Japan. In the present study, we conducted a survey of kudoid infections in tunas (the yellowfin tuna Thunnus albacares, the Pacific bluefin tuna Thunnus orientalis, and the longtail tuna Thunnus tonggol) fished in the western Pacific Ocean off Japan and several East Asian countries and characterized morphologically and genetically the kudoid myxospores in pseudocysts or cysts dispersed in the trunk muscles. Pseudocysts of solely Kudoa hexapunctata were identified in the Pacific bluefin tuna (four isolates), whereas in the yellowfin tuna (21 isolates) pseudocysts of Kudoa neothunni and K. hexapunctata were detected at a ratio of 15:6, respectively, in addition to cyst-forming Kudoa thunni in five yellowfin tunas. In the trunk muscles of six longtail tunas examined, pseudocysts of K. neothunni (all six fish) and K. hexapunctata (two fish) were densely dispersed. The myxospores of K. neothunni found in these longtail tunas had seven shell valves and polar capsules (SV/PC) instead of the more common six SV/PC arranged symmetrically. Nucleotide sequences of the 18S and 28S ribosomal RNA gene (rDNA), some with the internal transcribed spacer regions as well, of K. hexapunctata and K. neothunni from the three Thunnus spp., including the seven-SV/PC morphotype, were very similar to previously characterized nucleotide sequences of each species, whereas the 18S and 28S rDNA of four isolates of K. thunni from yellowfin tunas showed a range of nucleotide variations of 99.0-99.9% identity over 1752-1763-bp long partial 18S rDNA and 97.4-99.9% identity over 797-802-bp long partial 28S rDNA. Therefore, this rather high variation of the rDNA nucleotide sequences of K. thunni proved to be contrary to the few variations of K. neothunni and K. hexapunctata rDNA nucleotide sequences. The present study provides a new host record of the longtail tuna for K. neothunni and K. hexapunctata and reveals a high prevalence of the seven-SV/PC myxospore morphotype of K. neothunni in this tuna host.

Molecular detection of Sarcocystis lutrae in the European badger (Meles meles) in Scotland.

PubMed

Lepore, T; Bartley, P M; Chianini, F; Macrae, A I; Innes, E A; Katzer, F

2017-09-01

Neck samples from 54 badgers and 32 tongue samples of the same badgers (Meles meles), collected in the Lothians and Borders regions of Scotland, were tested using polymerase chain reactions (PCRs) directed against the 18S ribosomal DNA and the internal transcribed spacer (ITS1) region of protozoan parasites of the family Sarcocystidae. Positive results were obtained from 36/54 (67%) neck and 24/32 (75%) tongue samples using an 18S rDNA PCR. A 468 base pair consensus sequence that was generated from the 18S rDNA PCR amplicons (KX229728) showed 100% identity to Sarcocystis lutrae. The ITS1 PCR results revealed that 12/20 (60%) neck and 10/20 (50%) tongue samples were positive for Sarcocystidae DNA. A 1074 bp consensus sequence was generated from the ITS1 PCR amplicons (KX431307) and showed 100% identity to S. lutrae. Multiple sequence alignments and phylogenetic analysis support the finding that the rDNA found in badgers is identical to that of S. lutrae. This parasite has not been previously reported in badgers or in the UK. Sarcocystis lutrae has previously only been detected in tongue, skeletal muscle and diaphragm samples of the Eurasian otter (Lutra lutra) in Norway and potentially in the Arctic fox (Vulpes lagopus).

[The use of 16S rDNA sequencing in species diversity analysis for sputum of patients with ventilator-associated pneumonia].

PubMed

Yang, Xiaojun; Wang, Xiaohong; Liang, Zhijuan; Zhang, Xiaoya; Wang, Yanbo; Wang, Zhenhai

2014-05-01

To study the species and amount of bacteria in sputum of patients with ventilator-associated pneumonia (VAP) by using 16S rDNA sequencing analysis, and to explore the new method for etiologic diagnosis of VAP. Bronchoalveolar lavage sputum samples were collected from 31 patients with VAP. Bacterial DNA of the samples were extracted and identified by polymerase chain reaction (PCR). At the same time, sputum specimens were processed for routine bacterial culture. The high flux sequencing experiment was conducted on PCR positive samples with 16S rDNA macro genome sequencing technology, and sequencing results were analyzed using bioinformatics, then the results between the sequencing and bacteria culture were compared. (1) 550 bp of specific DNA sequences were amplified in sputum specimens from 27 cases of the 31 patients with VAP, and they were used for sequencing analysis. 103 856 sequences were obtained from those sputum specimens using 16S rDNA sequencing, yielding approximately 39 Mb of raw data. Tag sequencing was able to inform genus level in all 27 samples. (2) Alpha-diversity analysis showed that sputum samples of patients with VAP had significantly higher variability and richness in bacterial species (Shannon index values 1.20, Simpson index values 0.48). Rarefaction curve analysis showed that there were more species that were not detected by sequencing from some VAP sputum samples. (3) Analysis of 27 sputum samples with VAP by using 16S rDNA sequences yielded four phyla: namely Acitinobacteria, Bacteroidetes, Firmicutes, Proteobacteria. With genus as a classification, it was found that the dominant species included Streptococcus 88.9% (24/27), Limnohabitans 77.8% (21/27), Acinetobacter 70.4% (19/27), Sphingomonas 63.0% (17/27), Prevotella 63.0% (17/27), Klebsiella 55.6% (15/27), Pseudomonas 55.6% (15/27), Aquabacterium 55.6% (15/27), and Corynebacterium 55.6% (15/27). (4) Pyrophosphate sequencing discovered that Prevotella, Limnohabitans, Aquabacterium, Sphingomonas might not be detected by routine bacteria culture. Among seven species which were identified by both methods, pyrophosphate sequencing yielded higher positive rate than that of ordinary bacteria culture [Streptococcus: 88.9% (24/27) vs. 18.5% (5/27), Klebsiella: 55.6% (15/27) vs. 18.5% (5/27), Acinetobacter: 70.4% (19/27) vs. 37.0% (10/27), Corynebacterium: 55.6% (15/27) vs. 7.4% (2/27), P<0.05 or P<0.01]. Sequencing positive rate was found to increase positive rate for culture of Pseudomonas [55.6% (15/27) vs. 25.9% (7/27), P=0.050]. No significant differences were observed between sequencing and ordinary bacteria culture for detection Staphylococcus [7.4% (2/27) vs. 11.1% (3/27)] and Neisseria bacteria genera [18.5% (5/27) vs. 3.7% (1/27), both P>0.05]. 16S rDNA sequencing analysis confirmed that pathogenic bacteria in sputum of VAP were complicated with multiple drug resistant strains. Compared with routine bacterial culture, pyrophosphate sequencing had higher positive rate in detecting pathogens. 16S rDNA gene sequencing technology may become a new method for etiological diagnosis of VAP.

Authentication of an endangered herb Changium smyrnioides from different producing areas based on rDNA ITS sequences and allele-specific PCR.

PubMed

Sun, Xiaoqin; Wei, Yanglian; Qin, Minjian; Guo, Qiaosheng; Guo, Jianlin; Zhou, Yifeng; Hang, Yueyu

2012-03-01

The rDNA ITS region of 18 samples of Changium smyrnioides from 7 areas and of 2 samples of Chuanminshen violaceum were sequenced and analyzed. The amplified ITS region of the samples, including a partial sequence of ITS1 and complete sequences of 5.8S and ITS2, had a total length of 555 bp. After complete alignment, there were 49 variable sites, of which 45 were informative, when gaps were treated as missing data. Samples of C. smyrnioides from different locations could be identified exactly based on the variable sites. The maximum parsimony (MP) and neighbor joining (NJ) tree constructed from the ITS sequences based on Kumar's two-parameter model showed that the genetic distances of the C. smyrnioides samples from different locations were not always related to their geographical distances. A specific primer set for Allele-specific PCR authentication of C. violaceum from Jurong of Jiangsu was designed based on the SNP in the ITS sequence alignment. C. violaceum from the major genuine producing area in Jurong of Jiangsu could be identified exactly and quickly by Allele-specific PCR.

Trichomonas vaginalis ribosomal RNA: identification and characterisation of the transcription promoter and terminator sequences.

PubMed

Franco, Bernardo; Hernández, Roberto; López-Villaseñor, Imelda

2012-09-01

Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180 bp. We also identified a potential upstream control element (UCE) that was located 80 bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34 bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism. Copyright © 2012 Elsevier B.V. All rights reserved.

Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.

PubMed

Singh, K M; Tripathi, A K; Pandya, P R; Rank, D N; Kothari, R K; Joshi, C G

2011-09-01

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.

Molecular identification of Trichuris vulpis and Trichuris suis isolated from different hosts.

PubMed

Cutillas, Cristina; de Rojas, Manuel; Ariza, Concepción; Ubeda, José Manuel; Guevara, Diego

2007-01-01

Trichuris suis was isolated from the cecum of two different hosts (Sus scrofa domestica -- swine and Sus scrofa scrofa -- wild boar) and Trichuris vulpis from dogs in Sevilla, Spain. Genomic DNA was isolated and internal transcribed spacers (ITS)1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The sequence of T. suis from both hosts was 1,396 bp in length while that of T. vulpis was 1,044 bp. ITS1 of both populations isolated of T. suis was 661 nucleotides in length, while the ITS2 was 534 nucleotides in length. Furthermore, the ITS1 of T. vulpis was 410 nucleotides in length, while the ITS2 was 433 nucleotides in length. One hundred fifty-four nucleotides were observed along the 5.8S gene of T. suis and T. vulpis. Intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all the individuals assayed. Sequence analysis of the ITSs and the 5.8S gene has demonstrated no sequence differences between T. suis isolated from both hosts (S. scrofa domestica -- swine and S. scrofa scrofa -- wild boar). Nevertheless, clear differences were detected between the ITS1 and ITS2 of T. suis and T. vulpis. Furthermore, a comparative molecular analysis between both species and the previously published ITS1-5.8S-ITS2 sequence data of Trichuris ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris skrjabini was carried out. A common homology zone was detected in the ITS1 sequence of all species of trichurids.

Mitochondrial DNA sequence-based phylogenetic relationship of Trichiurus lepturus (Perciformes: Trichiuridae) from the Persian Gulf

PubMed Central

Tamadoni Jahromi, S.; Mohd Noor, S. A.; Pirian, K.; Dehghani, R.; Nazemi, M.; Khazaali, A.

2016-01-01

In this study, mitochondrial DNA analysis using 16S ribosomal DNA (rDNA) was performed to investigate the phylogeny relationship of Trichiurus lepturus in the Persian Gulf compared to the other investigated area. The amplification of 16S rDNA resulted in a product of 600 bp in all samples. The results showed that the isolated strain belongs to T. lepturus showing 42 divergence sites among the same reported partial sequences of 16S rRNA gene from the other area (West Atlantic and Indo-Pacific area). Phylogeny results showed that all 18 haplotypes of the species clustered into five clades with reasonably high bootstrap support of values (>64%). Overall, the tree topology for both phylogenetic and phenetic trees for 16S rDNA was similar. Both trees exposed two major clusters, one wholly containing the haplotypes of the T. lepturus species belonging to Indo-Pacific area with two major sister groups including Persian Gulf specimen and the other cleared the Western Atlantic and Japan individuals clustered in another distinct clade supporting the differentiation between the two areas. Phylogenic relationship observed between the Persian Gulf and the other Indo-Pacific Individuals suggested homogeneity between two mentioned areas. PMID:27822250

Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

PubMed

Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

2013-11-01

Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai.

Characterization of Microbial Communities in Gas Industry Pipelines

PubMed Central

Zhu, Xiang Y.; Lubeck, John; Kilbane, John J.

2003-01-01

Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines. Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed. A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of Proteobacteria and gram-positive bacteria. The most frequently encountered bacterial species was Comamonas denitrificans, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion. The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I, Methanobacteriales; order III, Methanomicrobiales; and order IV, Methanosarcinales. Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion. PMID:12957923

Two myxozoans from the urinary tract of topsmelt, Atherinops affinis

USGS Publications Warehouse

Sanders, Justin L.; Jaramillo, Alejandra G.; Ashford, Jacob E.; Feist, Stephen W.; Lafferty, Kevin D.; Kent, Michael L.

2015-01-01

Two myxozoan species were observed in the kidney of topsmelt, Atherinops affinis, during a survey of parasites of estuarine fishes in the Carpinteria Salt Marsh Reserve, California. Fish collected on three dates in 2012 and 2013 were sectioned and examined histologically. Large extrasporogonic stages occurred in the renal interstitium of several fish from the first two collections (5/8, 11/20, respectively), and, in some fish, these replaced over 80% of the kidney. In addition, presporogonic and polysporogonic stages occurred in the lumen of the renal tubules, collecting and mesonephric ducts. The latter contained subspherical spores with up to 4 polar capsules, consistent with the genus Chloromyxum. For the third collection (15 May 2013, n=30), we portioned kidneys for examination by histology, wet mount, and DNA extraction for small subunit ribosomal gene sequencing. Histology showed the large extrasporogonic forms in the kidney interstitium of 3 fish, and 2 other fish with subspherical myxospores in the lumen of the renal tubules with smooth valves and two spherical polar capsules consistent with the genus Sphaerospora. Chloromyxum-type myxospores were observed in the renal tubules of one fish by wet mount. Sequencing of the kidney tissue from this fish yielded a partial SSU rDNA sequence of 1769 bp. Phylogenetic reconstruction suggested this organism to be a novel species of Chloromyxum, most similar to Chloromyxum careni (84% similarity). In addition, subspherical myxospores with smooth valves and two spherical polar capsules consistent with the genus Sphaerospora were observed in wet mounts of 2 fish. Sequencing of the kidney tissue from 1 fish yielded a partial SSU rDNA sequence of 1937 bp. Phylogenetic reconstruction suggests this organism to be a novel species of Sphaerospora most closely related to Sphaerospora epinepheli (93%). We conclude that these organisms represent novel species of the genera Chloromyxum and Sphaerospora based on host, location, and SSU rDNA sequence. We further conclude that the formation of large, histozoic extrasprogonic stages in the renal interstitium represent developmental stages of the Chloromyxum species for the following reasons: 1. Large extrasporogonic stages stages were only observed in fish with Chloromyxum-type spores developing within the renal tubules, 2. DNA sequence consistent with the Chloromyxum sp. was only detected in fish with the large extrasporogonic stages and 3.Sphaerospora species have extrasporogonic forms, but they are considerably smaller and are comprised of much fewer cells.

Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

PubMed

Teixeira, M M; Campaner, M; Camargo, E P

1994-01-01

To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia).

PubMed

Kawagoshi, Taiki; Nishida, Chizuko; Ota, Hidetoshi; Kumazawa, Yoshinori; Endo, Hideki; Matsuda, Yoichi

2008-01-01

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30-42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG)( n ) sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.

Malassezia furfur in infantile seborrheic dermatitis.

PubMed

Wananukul, Siriwan; Chindamporn, Ariya; Yumyourn, Poomjit; Payungporn, Sunchai; Samathi, Chanchuree; Poovorawan, Yong

2005-01-01

Our objective was to study both incidence and various strains of Malassezia in infantile seborrheic dermatitis (ISD). Sixty infants between 2 weeks and 2 years old with clinical diagnosis of ISD at the Department of Pediatrics, King Chulalongkorn Memorial Hospital from May 2002 to April 2003 were recruited. Malassezia spp. were isolated from cultured skin samples of the patients, genomic DNA was extracted and the ITS1 rDNA region was amplified. The PCR product was examined by agarose gel electrophoresis and DNA sequences were determined. The ITS1 sequences were also subjected to phylogenetic analysis and species identification. ISD is most commonly found in infants below the age of 2 months (64%), followed by those between 2 and 4 months (28%) old. Cultures yielded yeast-like colonies in 15 specimens. PCR yielded 200-bp products (Candida) in 3 patients and 300-bp products (Malassezia furfur) in 12 patients (18%). Sugar fermentation using API 20C aux performed on the three 200-bp PCR products yielded Candida species. M. furfur was the only Malassezia recovered from skin scrapings of children with ISD.

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

PubMed

van Keulen, H; Gutell, R R; Campbell, S R; Erlandsen, S L; Jarroll, E L

1992-10-01

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan.

PubMed

Tigani-Asil, A E El; Sanousi, S M El; Aljameel, M A; Beir, H El; Adam, A; Abdallatif, M M; Hamid, M E

2014-01-01

This study identified nontuberculous mycobacteria (NTM) recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB) using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32) isolates, 550 bp. (2) isolates and 600 bp. (14) isolates. Four isolates were contaminated.

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

PubMed Central

Matsuda, M; Tazumi, A; Kagawa, S; Sekizuka, T; Murayama, O; Moore, JE; Millar, BC

2006-01-01

Background At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Results Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. Conclusion High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted. PMID:16398935

Vertical transmission of Theileria lestoquardi in sheep.

PubMed

Zakian, Amir; Nouri, Mohammad; Barati, Farid; Kahroba, Hooman; Jolodar, Abbas; Rashidi, Fardokht

2014-07-14

This is the first report of an outbreak of Theileria lestoquardi abortion and stillbirth in a mob of 450 ewes in July 2012, during which, approximately 35 late-term ewes lost their fetuses over a 5-day period. A dead ewe and her aborted fetus were transported to the Ahvaz Veterinary Hospital for the diagnostic evaluation. The microbial cultures from the ewe vaginal discharges and fetal abomasal contents and the liver were negative. The blood films of the ewe and her fetus contained Theileria piroplasms and the impression smears from ewe liver and fetal spleen were positive for Theileria Koch blue bodies. The DNA was extracted from the liver and spleen of ewe and her fetus, respectively, and analyzed by polymerase chain reaction (PCR) using specific primers derived from the nucleotide sequences of 18S ribosomal DNA (rDNA) gene of T. lestoquardi. A single fragment of 428-bp fragment was amplified. The PCR product was directly sequenced and the alignment of the sequence with similar sequences in GenBank(®) showed 100% identities with 18S rDNA gene of T. lestoquardi. The present study is the first report of the T. lestoquardi vertical transmission that could be related to the abortion. Copyright © 2014 Elsevier B.V. All rights reserved.

Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

PubMed

El-Sherry, Shiem; Ogedengbe, Mosun E; Hafeez, Mian A; Barta, John R

2013-07-01

Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks. Copyright © 2013. Published by Elsevier Ltd.

Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus.

PubMed

Chun, J; Huq, A; Colwell, R R

1999-05-01

Vibrio cholerae identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely related Vibrio mimicus. The two species share many genes coding for proteins, such as ctxAB, and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNAGlu, tRNALys, and tRNAVal, which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNAIle and tDNAAla, whereas the 500-bp fragment had single tDNA coding either tRNAGlu or tRNAAla. Little variation, i.e., 0 to 0.4%, was found among V. cholerae O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) and V. mimicus (2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all of V. cholerae strains from V. mimicus. The PCR system developed was subsequently evaluated by using representatives of V. cholerae from environmental and clinical sources, and of other taxa, including V. mimicus. This study provides the first molecular tool for identifying the species V. cholerae.

Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan

PubMed Central

Tigani-Asil, A.E. El; Sanousi, S.M. EL; Aljameel, M.A.; Beir, H. El; Adam, A.; Abdallatif, M.M.; Hamid, M.E.

2014-01-01

This study identified nontuberculous mycobacteria (NTM) recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB) using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32) isolates, 550 bp. (2) isolates and 600 bp. (14) isolates. Four isolates were contaminated. PMID:26623334

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina).

PubMed

Dentinger, Bryn T M; Didukh, Maryna Y; Moncalvo, Jean-Marc

2011-01-01

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.

Comparing COI and ITS as DNA Barcode Markers for Mushrooms and Allies (Agaricomycotina)

PubMed Central

Dentinger, Bryn T. M.; Didukh, Maryna Y.; Moncalvo, Jean-Marc

2011-01-01

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (∼450 bp) representing ∼100 morphospecies from ∼650 collections of Agaricomycotina using several sets of new primers. Large introns (∼1500 bp) at variable locations were detected in ∼5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (∼30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms. PMID:21966418

Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome.

PubMed

Robicheau, Brent M; Susko, Edward; Harrigan, Amye M; Snyder, Marlene

2017-02-01

Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria.

PubMed

Loong, Shih Keng; Khor, Chee Sieng; Jafar, Faizatul Lela; AbuBakar, Sazaly

2016-11-01

Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5-year study period, with special emphasis on isolates that gave unsatisfactory identification. One hundred and eighty-seven clinical bacteria isolates were tested with commercial phenotypic identification systems and 16S rDNA sequencing. Isolate identities determined using phenotypic identification systems and 16S rDNA sequencing were compared for similarity at genus and species level, with 16S rDNA sequencing as the reference method. Phenotypic identification systems identified ~46% (86/187) of the isolates with identity similar to that identified using 16S rDNA sequencing. Approximately 39% (73/187) and ~15% (28/187) of the isolates showed different genus identity and could not be identified using the phenotypic identification systems, respectively. Both methods succeeded in determining the species identities of 55 isolates; however, only ~69% (38/55) of the isolates matched at species level. 16S rDNA sequencing could not determine the species of ~20% (37/187) of the isolates. The 16S rDNA sequencing is a useful method over the phenotypic identification systems for the identification of rare and difficult to identify bacteria species. The 16S rDNA sequencing method, however, does have limitation for species-level identification of some bacteria highlighting the need for better bacterial pathogen identification tools. © 2016 Wiley Periodicals, Inc.

Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

PubMed

Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

2016-09-01

Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species.

Nanobacteria may be linked to calcification in placenta.

PubMed

Lu, He; Guo, Ya-nan; Liu, Sheng-nan; Zhang, De-chun

2012-05-01

Placental calcification is a common pathologic condition in obstetrics. To detect the bacteria infection mechanisms for calcification, an experiment was performed to isolate, culture, and identify the nanobacteria in placental calcification. Sixteen cases of placental calcification of pregnant women were collected for the purpose of the isolation of nanobacteria, cultivation, and identification of 16S rDNA sequence. Under transmission electron microscope, novel oval-shape nanobacteria-like particles (NLP) in extracellular matrix of calcified placenta tissues were found with 50-500 nm in diameter, and among hydroxyapatite crystals aggregation existed. After about 4 weeks of culturing and isolating NLP from these calcified tissues, all calcified placental tissue samples and one adjacent tissue of calcified placental tissue samples showed white granular depositions, which were firmly attached to the bottom of the culture tubes and visible to the naked eyes. In the control group they could not be seen. After PCR was amplified a 1407-bp fragment was obtained and submitted to GenBank after sequencing with accession number JN029830. The 16S rDNA sequence homology between the isolation strain and strain nanobacteria (X98418) was 92% in GenBank. For the first time isolated, cultured, and identified nanobacteria in placental calcification indicated that nanobacteria infection is related to placental calcification.

Intraspecific variability of Steinernema feltiae strains from Cemoro Lawang, eastern Java, Indonesia.

PubMed

Addis, T; Mulawarman, M; Waeyenberge, L; Moens, M; Viaene, N; Ehlers, R U

2010-01-01

Four strains of Steinernema feltiae from Eastern Java, Indonesia were characterized based on morphometric, morphological and molecular data. In addition, their virulence against last instar Tenebrio molitor and heat tolerance was tested. Infective juvenile have a mean body length ranging from 749 to 792 microm. The maximum sequence difference among the four strains was 7 bp (8.8%) in the ITS and 2 bp (0.3%) in D2D3 regions of the rDNA. All the strains are not reproductively isolated and can reproduce with European strain S. feltiae Owiplant. The lowest LC50 was observed for strain SCM (373) and the highest for S. feltiae strain Owiplant (458) IJs/40 T. molitor. All four strains showed relatively better mean heat tolerance when compared with S. feltiae Owiplant, both in adapted and non-adapted heat tolerance experiments.

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

PubMed Central

Rölleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

1996-01-01

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

Fragile sites, dysfunctional telomere and chromosome fusions: What is 5S rDNA role?

PubMed

Barros, Alain Victor; Wolski, Michele Andressa Vier; Nogaroto, Viviane; Almeida, Mara Cristina; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo

2017-04-15

Repetitive DNA regions are known as fragile chromosomal sites which present a high flexibility and low stability. Our focus was characterize fragile sites in 5S rDNA regions. The Ancistrus sp. species shows a diploid number of 50 and an indicative Robertsonian fusion at chromosomal pair 1. Two sequences of 5S rDNA were identified: 5S.1 rDNA and 5S.2 rDNA. The first sequence gathers the necessary structures to gene expression and shows a functional secondary structure prediction. Otherwise, the 5S.2 rDNA sequence does not contain the upstream sequences that are required to expression, furthermore its structure prediction reveals a nonfunctional ribosomal RNA. The chromosomal mapping revealed several 5S.1 and 5S.2 rDNA clusters. In addition, the 5S.2 rDNA clusters were found in acrocentric and metacentric chromosomes proximal regions. The pair 1 5S.2 rDNA cluster is co-located with interstitial telomeric sites (ITS). Our results indicate that its clusters are hotspots to chromosomal breaks. During the meiotic prophase bouquet arrangement, double strand breaks (DSBs) at proximal 5S.2 rDNA of acrocentric chromosomes could lead to homologous and non-homologous repair mechanisms as Robertsonian fusions. Still, ITS sites provides chromosomal instability, resulting in telomeric recombination via TRF2 shelterin protein and a series of breakage-fusion-bridge cycles. Our proposal is that 5S rDNA derived sequences, act as chromosomal fragile sites in association with some chromosomal rearrangements of Loricariidae. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of a third feline Demodex species through partial sequencing of the 16S rDNA and frequency of Demodex species in 74 cats using a PCR assay.

PubMed

Ferreira, Diana; Sastre, Natalia; Ravera, Iván; Altet, Laura; Francino, Olga; Bardagí, Mar; Ferrer, Lluís

2015-08-01

Demodex cati and Demodex gatoi are considered the two Demodex species of cats. However, several reports have identified Demodex mites morphologically different from these two species. The differentiation of Demodex mites is usually based on morphology, but within the same species different morphologies can occur. DNA amplification/sequencing has been used effectively to identify and differentiate Demodex mites in humans, dogs and cats. The aim was to develop a PCR technique to identify feline Demodex mites and use this technique to investigate the frequency of Demodex in cats. Demodex cati, D. gatoi and Demodex mites classified morphologically as the third unnamed feline species were obtained. Hair samples were taken from 74 cats. DNA was extracted; a 330 bp fragment of the 16S rDNA was amplified and sequenced. The sequences of D. cati and D. gatoi shared >98% identity with those published on GenBank. The sequence of the third unnamed species showed 98% identity with a recently published feline Demodex sequence and only 75.2 and 70.9% identity with D. gatoi and D. cati sequences, respectively. Demodex DNA was detected in 19 of 74 cats tested; 11 DNA sequences corresponded to Demodex canis, five to Demodex folliculorum, three to D. cati and two to Demodex brevis. Three Demodex species can be found in cats, because the third unnamed Demodex species is likely to be a distinct species. Apart from D. cati and D. gatoi, DNA from D. canis, D. folliculorum and D. brevis was found on feline skin. © 2015 ESVD and ACVD.

[Sequence of the ITS region of nuclear ribosomal DNA(nrDNA) in Xinjiang wild Dianthus and its phylogenetic relationship].

PubMed

Zhang, Lu; Cai, You-Ming; Zhuge, Qiang; Zou, Hui-Yu; Huang, Min-Ren

2002-06-01

Xinjiang is a center of distribution and differentiation of genus Dianthus in China, and has a great deal of species resources. The sequences of ITS region (including ITS-1, 5.8S rDNA and ITS-2) of nuclear ribosomal DNA from 8 species of genus Dianthus wildly distributed in Xinjiang were determined by direct sequencing of PCR products. The result showed that the size of the ITS of Dianthus is from 617 to 621 bp, and the length variation is only 4 bp. There are very high homogeneous (97.6%-99.8%) sequences between species, and about 80% homogeneous sequences between genus Dianthus and outgroup. The sequences of ITS in genus Dianthus are relatively conservative. In general, there are more conversion than transition in the variation sites among genus Dianthus. The conversion rates are relatively high, and the ratios of conversion/transition are 1.0-3.0. On the basis of phylogenetic analysis of nucleotide sequences the species of Dianthus in China would be divided into three sections. There is a distant relationship between sect. Barbulatum Williams and sect. Dianthus and between sect. Barbulatum Williams and sect. Fimbriatum Williams, and there is a close relationship between sect. Dianthus and sect. Fimbriatum Williams. From the phylogenetic tree of ITS it was found that the origin of sect. Dianthusis is earlier than that of sect. Fimbriatum Williams and sect. Barbulatum Williams.

ITS2 as a molecular marker for the identification of Diatraea saccharalis and D. flavipennella and possible infection with Cotesia spp.

PubMed

Neto, M S Regueira; Barros, R P; Morais, M A J; Balbino, V Q; Loreto, V

2017-08-31

In Brazil, the species Diatraea flavipennella and D. saccharalis play an important role in the sugar and alcohol agribusiness by causing many damages in sugarcane fields. The egg, larva, pupa, and adult stages are very morphologically similar between these species, and the identification can be confused. The internal transcribed spacer 2 (ITS 2) from ribosomal DNA has important features as evolutionary divergence. It is a good marker for species identification, participates in the rDNA processing, and has been applied in phylogenetic and population studies. This study aimed to make available a molecular marker to assist on the identification method of pests' species of Diatraea and to identify possible traces of Cotesia in the resistant host. The DNA was extracted from the egg, larva, and adult samples. PCR amplicons were purified and sequenced. The sequences were analyzed in MEGA 5.01. The ITS 2 length was 410 bp in D. flavipennella and 448 bp in D. saccharalis. The GC content was similar between the species. Three microsatellite loci were present in D. saccharalis and absent in D. flavipennella, contributing to differences in ITS 2 length in the species. An additional 367-bp band was attributed to Cotesia spp. The differences among ITS 2 from D. flavipennella, D. saccharalis, and Cotesia sp were sufficient to identify them on electrophoresis gel and sequencing. The presence of Cotesia sp traits in adult D. flavipennella showed possible host refractoriness, but further studies are necessary.

Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

PubMed Central

Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

2016-01-01

Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

Genetic differentiation of Artyfechinostomum malayanum and A. sufrartyfex (Trematoda: Echinostomatidae) based on internal transcribed spacer sequences.

PubMed

Tantrawatpan, Chairat; Saijuntha, Weerachai; Sithithaworn, Paiboon; Andrews, Ross H; Petney, Trevor N

2013-01-01

Genetic differentiation between two synonymous echinostomes species, Artyfechinostomum malayanum and Artyfechinostomum sufrartyfex was determined by using the first and second internal transcribed spacers (ITS1 and ITS2), the non-coding region of rDNA as genetic makers. Of the 699 bp of combined ITS1 and ITS2 sequences examined, 18 variable nucleotide positions (2.58 %) were observed. Of these, 17 positions could be used as diagnostic position between these two sibling species, whereas the other one variation was intraspecific variation of A. malayanum. A clade of A. malayanum was closely aligned with A. sufrartyfex and clearly distance from the cluster of other echinostomes. Our results may sufficiently suggest that the current synonymy of these species is not valid.

Genetic and morphological characterization of Trichuris myocastoris found in Myocastor coypus in the Czech Republic.

PubMed

Rylková, K; Tůmová, E; Brožová, A; Jankovská, I; Vadlejch, J; Čadková, Z; Frýdlová, J; Peřinková, P; Langrová, I; Chodová, D; Nechybová, S; Scháňková, Š

2015-11-01

Trichuris sp. individuals were collected from Myocastor coypus from fancy breeder farms in the Czech Republic. Using morphological and biometrical methods, 30 female and 30 male nematodes were identified as Trichuris myocastoris. This paper presents the first molecular description of this species. The ribosomal DNA (rDNA) region, consisting of internal transcribed spacer (ITS)-1, 5.8 gene and ITS-2, was sequenced. Based on an analysis of 651 bp, T. myocastoris was found to be different from any other Trichuris species for which published sequencing of the ITS region is available. The phylogenetic relationships were estimated using the maximum parsimony methods and Bayesian analyses. T. myocastoris was found to be significantly closely related to Trichuris of rodents than those of ruminants.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

PubMed

Volkov, Roman A; Panchuk, Irina I; Borisjuk, Nikolai V; Hosiawa-Baranska, Marta; Maluszynska, Jolanta; Hemleben, Vera

2017-01-23

Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Characterization of North American Armillaria species: Genetic relationships determined by ribosomal DNA sequences and AFLP markers

Treesearch

M. -S. Kim; N. B. Klopfenstein; J. W. Hanna; G. I. McDonald

2006-01-01

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data,...

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

Treesearch

M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

2009-01-01

The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

Phylogenetic assessment of heterotrophic bacteria from a water distribution system using 16S rDNA sequencing.

PubMed

Tokajian, Sima T; Hashwa, Fuad A; Hancock, Ian C; Zalloua, Pierre A

2005-04-01

Determination of a heterotrophic plate count (HPC) for drinking-water samples alone is not enough to assess possible health hazards associated with sudden changes in the bacterial count. Speciation is very crucial to determine whether the population includes pathogens and (or) opportunistic pathogens. Most of the isolates recovered from drinking water samples could not be allocated to a specific phylogenetic branch based on the use of conventional diagnostic methods. The present study had to use phylogenetic analysis, which was simplified by determining and using the first 500-bp sequence of the 16S rDNA, to successfully identify the type and species of bacteria found in the samples. Gram-positive bacteria alpha-, beta-, and gamma-Proteobacteria were found to be the major groups representing the heterotrophic bacteria in drinking water. The study also revealed that the presence of sphingomonads in drinking water supplies may be much more common than has been reported so far and thus further studies are merited. The intermittent mode of supply, mainly characterized by water stagnation and flow interruption associated possibly with biofilm detachment, raised the possibility that the studied bacterial populations in such systems represented organisms coming from 2 different niches, the biofilm and the water column.

Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences.

PubMed

Yong, T S; Park, S J; Hwang, U W; Yang, H W; Lee, K W; Min, D Y; Rim, H J; Wang, Y; Zheng, F

2000-08-01

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection.

PubMed

Perina, Alejandra; Seoane, David; González-Tizón, Ana M; Rodríguez-Fariña, Fernanda; Martínez-Lage, Andrés

2011-10-17

The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection

PubMed Central

2011-01-01

Background The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. Results The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. Conclusions These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection. PMID:22004418

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

Genetic and Biochemical Characterization of Monokaryotic Progeny Strains of Button Mushroom (Agaricus bisporus)

PubMed Central

Kwon, Hyuk Woo; Choi, Min Ah; Yun, Yeo Hong; Oh, Youn-Lee; Kong, Won-Sik

2015-01-01

To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (β-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains. PMID:25892920

Nuclear 28S rDNA phylogeny supports the basal placement of Noctiluca scintillans (Dinophyceae; Noctilucales) in dinoflagellates.

PubMed

Ki, Jang-Seu

2010-05-01

Noctiluca scintillans (Macartney) Kofoid et Swezy, 1921 is an unarmoured heterotrophic dinoflagellate with a global distribution, and has been considered as one of the ancestral taxa among dinoflagellates. Recently, 18S rDNA, actin, alpha-, beta-tubulin, and Hsp90-based phylogenies have shown the basal position of the noctilucids. However, the relationships of dinoflagellates in the basal lineages are still controversial. Although the nuclear rDNA (e.g. 18S, ITS-5.8S, and 28S) contains much genetic information, DNA sequences of N. scintillans rDNA molecules were insufficiently characterized as yet. Here the author sequenced a long-range nuclear rDNA, spanning from the 18S to the D5 region of the 28S rDNA, of N. scintillans. The present N. scintillans had a nearly identical genotype (>99.0% similarity) compared to other Noctiluca sequences from different geographic origins. Nucleotide divergence in the partial 28S rDNA was significantly high (p<0.05) as compared to the 18S rDNA, demonstrating that the information from 28S rDNA is more variable. The 28S rDNA phylogeny of 17 selected dinoflagellates, two perkinsids, and two apicomplexans as outgroups showed that N. scintillans and Oxyrrhis marina formed a clade that diverged separately from core dinoflagellates. Copyright (c) 2009 Elsevier GmbH. All rights reserved.

Evolution of rDNA in Nicotiana Allopolyploids: A Potential Link between rDNA Homogenization and Epigenetics

PubMed Central

Kovarik, Ales; Dadejova, Martina; Lim, Yoong K.; Chase, Mark W.; Clarkson, James J.; Knapp, Sandra; Leitch, Andrew R.

2008-01-01

Background The evolution and biology of rDNA have interested biologists for many years, in part, because of two intriguing processes: (1) nucleolar dominance and (2) sequence homogenization. We review patterns of evolution in rDNA in the angiosperm genus Nicotiana to determine consequences of allopolyploidy on these processes. Scope Allopolyploid species of Nicotiana are ideal for studying rDNA evolution because phylogenetic reconstruction of DNA sequences has revealed patterns of species divergence and their parents. From these studies we also know that polyploids formed over widely different timeframes (thousands to millions of years), enabling comparative and temporal studies of rDNA structure, activity and chromosomal distribution. In addition studies on synthetic polyploids enable the consequences of de novo polyploidy on rDNA activity to be determined. Conclusions We propose that rDNA epigenetic expression patterns established even in F1 hybrids have a material influence on the likely patterns of divergence of rDNA. It is the active rDNA units that are vulnerable to homogenization, which probably acts to reduce mutational load across the active array. Those rDNA units that are epigenetically silenced may be less vulnerable to sequence homogenization. Selection cannot act on these silenced genes, and they are likely to accumulate mutations and eventually be eliminated from the genome. It is likely that whole silenced arrays will be deleted in polyploids of 1 million years of age and older. PMID:18310159

Molecular analysis of microbiota along the digestive tract of juvenile Atlantic salmon (Salmo salar L.).

PubMed

Navarrete, P; Espejo, R T; Romero, J

2009-04-01

Dominant bacterial microbiota of the gut of juvenile farmed Atlantic salmon was investigated using a combination of molecular approaches. Bacterial community composition from the stomach, the pyloric caeca, and the intestine was assessed by extracting DNA directly from each gut compartment. Temporal temperature gradient gel electrophoresis (TTGE) analysis of 16S ribosomal DNA (rDNA) amplicons showed very similar bacterial compositions throughout the digestive tract. Band sequencing revealed a narrow diversity of species with a dominance of Pseudomonas in the three compartments. However, cloning revealed more diversity among the Pseudomonas sequences. To confirm these results, we analyzed the bacterial community by amplifying the variable 16S-23S rDNA intergenic spacer region (ITS). Similar ITS profiles were observed among gastrointestinal compartments of salmon, confirming the TTGE results. Moreover, the dominant ITS band at 650 bp, identified as Pseudomonas, was observed in the ITS profile from fish collected in two seasons (July 2003 and 2004). In contrast, aerobic culture analysis revealed Shewanella spp. as the most prevalent isolate. This discrepancy was resolved by evaluating 16S rDNA and ITS polymerase chain reaction amplification efficiency from both Shewanella and Pseudomonas isolates. Very similar efficiencies were observed in the two bacteria. Hence, this discrepancy may be explained by preferential cultivation of Shewanella spp. under the experimental conditions. Also, we included analyses of pelleted feed and the water influent to explore environmental influences on the bacterial composition of the gut microbiota. Overall, these results indicate a homogeneous composition of the bacterial community composition along the gastrointestinal tract of reared juvenile salmon. This community is mainly composed of Pseudomonas spp., which could be derived from water influent and may be selectively associated with salmon in this hatchery.

Variation of 45S rDNA intergenic spacers in Arabidopsis thaliana.

PubMed

Havlová, Kateřina; Dvořáčková, Martina; Peiro, Ramon; Abia, David; Mozgová, Iva; Vansáčová, Lenka; Gutierrez, Crisanto; Fajkus, Jiří

2016-11-01

Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5 % of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.

Molecular characterization and phylogenetic analysis of the causative agent of hemoplasma infection in small Indian Mongoose (Herpestes Javanicus).

PubMed

Sharifiyazdi, Hassan; Nazifi, Saeed; Shirzad Aski, Hesamaddin; Shayegh, Hossein

2014-09-01

Hemoplasmas are the trivial name for a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. This study is the first report of hemoplasma infection in Small Indian Mongoose (Herpestes Javanicus) based on molecular analysis of 16S rDNA. Whole blood samples were collected by sterile methods, from 14 live captured mongooses, in the south of Iran. Candidatus Mycoplasma turicensis (CMt)-like hemoplasma was detected in blood samples from one animal tested. BLAST search and phylogenetic analysis of partial 16S rDNA sequence (933bp) of the hemoplasma from Small Indian mongoose (KJ530704) revealed only 96-97% identity to the previously described CMt followed by 95% and 91% similarity with Mycoplasma coccoides and Mycoplasma haemomuris, respectively. Accordingly, the Iranian mongoose CMt isolate showed a high intra-specific genetic variation compared to all previously reported CMt strains in GenBank. Further molecular studies using multiple phylogenetic markers are required to characterize the exact species of Mongoose-derived hemoplasma. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses.

PubMed

Cheng, Tian-Fan; Jia, Xiao-Ming; Ma, Xiao-Hang; Lin, Hai-Ping; Zhao, Yu-Hua

2004-01-01

In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae. Copyright 2004 WILEY-VCH Verlag GmbH & Co.

Phylogenetic tree of 16s rRNA sequences from sulfate-reducing bacteria in a sandy marine sediment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devereux, R.; Mundfrom, G.W.

1994-01-01

Phylogenetic divergence among sulfate-reducing bateria in an estuarine sediment sample was investigated by PCR amplification and comparison of partial 16S rDNA sequences. Twenty unique 16S rDNA sequences were found, 12 from delta subclass bacteria based on overall sequence similarity (82-91%). Two successive PCR amplifications were used to obtain and clone the 16S rDNA. The first reaction used templates derived from phosphate-buffered saline washed sediment with primers designed to amplify nearly full-length bacterial domain 16S rDNA. A produce from a first reaction was used as template in a second reaction with primers designed to selectivity amplify a region of 16S rDNAmore » genes of sulfate-reducing bacteria. A phylogenetic tree incorporating the cloned sequences suggests the presence of yet to be cultivated lines of sulfate-reducing bacteria within the sediment sample.« less

Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera.

PubMed

Campo, Daniel; García-Vázquez, Eva

2012-01-01

The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex

PubMed Central

Chand Dakal, Tikam; Giudici, Paolo; Solieri, Lisa

2016-01-01

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding. PMID:27501051

Molecular Characterization of Lactobacillus plantarum DMDL 9010, a Strain with Efficient Nitrite Degradation Capacity

PubMed Central

Fei, Yong-tao; Liu, Dong-mei; Luo, Tong-hui; Chen, Gu; Wu, Hui; Li, Li; Yu, Yi-gang

2014-01-01

Nitrites commonly found in food, especially in fermented vegetables, are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. A Lactobacillus strain (Lactobacillus sp. DMDL 9010) was previously isolated from fermented vegetables by our group, and is not yet fully characterized. A number of phenotypical and genotypical approaches were employed to characterize Lactobacillus sp. DMDL 9010. Its nitrite degradation capacity was compared with four other Lactobacillus strains, including Lactobacillus casei subsp. rhamnosus 719, Lactobacillus delbrueckii subsp. bulgaricu 1.83, Streptococcus thermophilus 1.204, and lactobacillus plantarum 8140, on MRS medium. Compared to these four Lactobacillus strains, Lactobacillus sp. DMDL 9010 had a significantly higher nitrite degradation capacity (P<0.001). Based on 16S rDNA sequencing and sequence comparison, Lactobacillus sp. DMDL 9010 was identified as either Lactobacillus plantarum or Lactobacillus pentosus. To further identify this strain, the flanking regions (922 bp and 806 bp upstream and downstream, respectively) of the L-lactate dehydrogenase 1 (L-ldh1) gene were amplified and sequenced. Lactobacillus sp. DMDL 9010 had 98.92 and 76.98% sequence identity in the upstream region with L. plantarum WCFS1 and L. pentosus IG1, respectively, suggesting that Lactobacillu sp. DMDL 9010 is an L. plantarum strain. It was therefore named L. plantarum DMDL 9010. Our study provides a platform for genetic engineering of L. plantarum DMDL 9010, in order to further improve its nitrite degradation capacity. PMID:25423449

Molecular characterization of Lactobacillus plantarum DMDL 9010, a strain with efficient nitrite degradation capacity.

PubMed

Fei, Yong-tao; Liu, Dong-mei; Luo, Tong-hui; Chen, Gu; Wu, Hui; Li, Li; Yu, Yi-gang

2014-01-01

Nitrites commonly found in food, especially in fermented vegetables, are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. A Lactobacillus strain (Lactobacillus sp. DMDL 9010) was previously isolated from fermented vegetables by our group, and is not yet fully characterized. A number of phenotypical and genotypical approaches were employed to characterize Lactobacillus sp. DMDL 9010. Its nitrite degradation capacity was compared with four other Lactobacillus strains, including Lactobacillus casei subsp. rhamnosus 719, Lactobacillus delbrueckii subsp. bulgaricu 1.83, Streptococcus thermophilus 1.204, and lactobacillus plantarum 8140, on MRS medium. Compared to these four Lactobacillus strains, Lactobacillus sp. DMDL 9010 had a significantly higher nitrite degradation capacity (P<0.001). Based on 16S rDNA sequencing and sequence comparison, Lactobacillus sp. DMDL 9010 was identified as either Lactobacillus plantarum or Lactobacillus pentosus. To further identify this strain, the flanking regions (922 bp and 806 bp upstream and downstream, respectively) of the L-lactate dehydrogenase 1 (L-ldh1) gene were amplified and sequenced. Lactobacillus sp. DMDL 9010 had 98.92 and 76.98% sequence identity in the upstream region with L. plantarum WCFS1 and L. pentosus IG1, respectively, suggesting that Lactobacillu sp. DMDL 9010 is an L. plantarum strain. It was therefore named L. plantarum DMDL 9010. Our study provides a platform for genetic engineering of L. plantarum DMDL 9010, in order to further improve its nitrite degradation capacity.

Application of Faecalibacterium 16S rDNA genetic marker for accurate identification of duck faeces.

PubMed

Sun, Da; Duan, Chuanren; Shang, Yaning; Ma, Yunxia; Tan, Lili; Zhai, Jun; Gao, Xu; Guo, Jingsong; Wang, Guixue

2016-04-01

The aim of this study was to judge the legal duty of pollution liabilities by assessing a duck faeces-specific marker, which can exclude distractions of residual bacteria from earlier contamination accidents. With the gene sequencing technology and bioinformatics method, we completed the comparative analysis of Faecalibacterium sequences, which were associated with ducks and other animal species, and found the sequences unique to duck faeces. Polymerase chain reaction (PCR) and agarose gel electrophoresis techniques were used to verify the reliability of both human and duck faeces-specific primers. The duck faeces-specific primers generated an amplicon of 141 bp from 43.3 % of duck faecal samples, 0 % of control samples and 100 % of sewage wastewater samples that contained duck faeces. We present here the initial evidence of Faecalibacterium-based applicability as human faeces-specificity in China. Meanwhile, this study represents the initial report of a Faecalibacterium marker for duck faeces and suggests an independent or supplementary environmental biotechnology of microbial source tracking (MST).

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

PubMed Central

Pan, W J; Blackburn, E H

1995-01-01

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number. Images PMID:7784211

[Structural organization of 5S ribosomal DNA of Rosa rugosa].

PubMed

Tynkevych, Iu O; Volkov, R A

2014-01-01

In order to clarify molecular organization of the genomic region encoding 5S rRNA in diploid species Rosa rugosa several 5S rDNA repeated units were cloned and sequenced. Analysis of the obtained sequences revealed that only one length variant of 5S rDNA repeated units, which contains intact promoter elements in the intergenic spacer region (IGS) and appears to be transcriptionally active is present in the genome. Additionally, a limited number of 5S rDNA pseudogenes lacking a portion of coding sequence and the complete IGS was detected. A high level of sequence similarity (from 93.7 to 97.5%) between the IGS of major 5S rDNA variants of East Asian R. rugosa and North American R. nitida was found indicating comparatively recent divergence of these species.

Blastocystis phylogeny among various isolates from humans to insects.

PubMed

Yoshikawa, Hisao; Koyama, Yukiko; Tsuchiya, Erika; Takami, Kazutoshi

2016-12-01

Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank. Copyright © 2016. Published by Elsevier Ireland Ltd.

The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

PubMed

Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Martí, Dardo Andrea; Mariguela, Tatiane Casagrande; Cabral-de-Mello, Diogo Cavalcanti

2016-08-01

The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.

Secondary structure prediction for complete rDNA sequences (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari.

PubMed

Zhao, Ya-E; Wang, Zheng-Hang; Xu, Yang; Wu, Li-Ping; Hu, Li

2013-10-01

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular Analysis of Dehalococcoides 16S Ribosomal DNA from Chloroethene-Contaminated Sites throughout North America and Europe

PubMed Central

Hendrickson, Edwin R.; Payne, Jo Ann; Young, Roslyn M.; Starr, Mark G.; Perry, Michael P.; Fahnestock, Stephen; Ellis, David E.; Ebersole, Richard C.

2002-01-01

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes. PMID:11823182

Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are gamma-radiation resistant

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Kempf, Michael; Chen, Fei; Satomi, Masataka; Nicholson, Wayne; Kern, Roger

2003-01-01

One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).

Mitochondrion-to-Chloroplast DNA Transfers and Intragenomic Proliferation of Chloroplast Group II Introns in Gloeotilopsis Green Algae (Ulotrichales, Ulvophyceae).

PubMed

Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-09-19

To probe organelle genome evolution in the Ulvales/Ulotrichales clade, the newly sequenced chloroplast and mitochondrial genomes of Gloeotilopsis planctonica and Gloeotilopsis sarcinoidea (Ulotrichales) were compared with those of Pseudendoclonium akinetum (Ulotrichales) and of the few other green algae previously sampled in the Ulvophyceae. At 105,236 bp, the G planctonica mitochondrial DNA (mtDNA) is the largest mitochondrial genome reported so far among chlorophytes, whereas the 221,431-bp G planctonica and 262,888-bp G sarcinoidea chloroplast DNAs (cpDNAs) are the largest chloroplast genomes analyzed among the Ulvophyceae. Gains of non-coding sequences largely account for the expansion of these genomes. Both Gloeotilopsis cpDNAs lack the inverted repeat (IR) typically found in green plants, indicating that two independent IR losses occurred in the Ulvales/Ulotrichales. Our comparison of the Pseudendoclonium and Gloeotilopsis cpDNAs offered clues regarding the mechanism of IR loss in the Ulotrichales, suggesting that internal sequences from the rDNA operon were differentially lost from the two original IR copies during this process. Our analyses also unveiled a number of genetic novelties. Short mtDNA fragments were discovered in two distinct regions of the G sarcinoidea cpDNA, providing the first evidence for intracellular inter-organelle gene migration in green algae. We identified for the first time in green algal organelles, group II introns with LAGLIDADG ORFs as well as group II introns inserted into untranslated gene regions. We discovered many group II introns occupying sites not previously documented for the chloroplast genome and demonstrated that a number of them arose by intragenomic proliferation, most likely through retrohoming. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mitochondrion-to-Chloroplast DNA Transfers and Intragenomic Proliferation of Chloroplast Group II Introns in Gloeotilopsis Green Algae (Ulotrichales, Ulvophyceae)

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-01-01

Abstract To probe organelle genome evolution in the Ulvales/Ulotrichales clade, the newly sequenced chloroplast and mitochondrial genomes of Gloeotilopsis planctonica and Gloeotilopsis sarcinoidea (Ulotrichales) were compared with those of Pseudendoclonium akinetum (Ulotrichales) and of the few other green algae previously sampled in the Ulvophyceae. At 105,236 bp, the G. planctonica mitochondrial DNA (mtDNA) is the largest mitochondrial genome reported so far among chlorophytes, whereas the 221,431-bp G. planctonica and 262,888-bp G. sarcinoidea chloroplast DNAs (cpDNAs) are the largest chloroplast genomes analyzed among the Ulvophyceae. Gains of non-coding sequences largely account for the expansion of these genomes. Both Gloeotilopsis cpDNAs lack the inverted repeat (IR) typically found in green plants, indicating that two independent IR losses occurred in the Ulvales/Ulotrichales. Our comparison of the Pseudendoclonium and Gloeotilopsis cpDNAs offered clues regarding the mechanism of IR loss in the Ulotrichales, suggesting that internal sequences from the rDNA operon were differentially lost from the two original IR copies during this process. Our analyses also unveiled a number of genetic novelties. Short mtDNA fragments were discovered in two distinct regions of the G. sarcinoidea cpDNA, providing the first evidence for intracellular inter-organelle gene migration in green algae. We identified for the first time in green algal organelles, group II introns with LAGLIDADG ORFs as well as group II introns inserted into untranslated gene regions. We discovered many group II introns occupying sites not previously documented for the chloroplast genome and demonstrated that a number of them arose by intragenomic proliferation, most likely through retrohoming. PMID:27503298

Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids

PubMed Central

2012-01-01

Background Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. Methods We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. Results and Conclusions In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations. PMID:23259460

The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

PubMed Central

Schwelm, Arne; Berney, Cédric; Dixelius, Christina; Bass, David; Neuhauser, Sigrid

2016-01-01

Clubroot disease caused by Plasmodiophora brassicae is one of the most important diseases of cultivated brassicas. P. brassicae occurs in pathotypes which differ in the aggressiveness towards their Brassica host plants. To date no DNA based method to distinguish these pathotypes has been described. In 2011 polymorphism within the 28S rDNA of P. brassicae was reported which potentially could allow to distinguish pathotypes without the need of time-consuming bioassays. However, isolates of P. brassicae from around the world analysed in this study do not show polymorphism in their LSU rDNA sequences. The previously described polymorphism most likely derived from soil inhabiting Cercozoa more specifically Neoheteromita-like glissomonads. Here we correct the LSU rDNA sequence of P. brassicae. By using FISH we demonstrate that our newly generated sequence belongs to the causal agent of clubroot disease. PMID:27750174

Phylogeny and genetic diversity of Bridgeoporus nobilissimus inferred using mitochondrial and nuclear rDNA sequences

USGS Publications Warehouse

Redberg, G.L.; Hibbett, D.S.; Ammirati, J.F.; Rodriguez, R.J.

2003-01-01

The genetic diversity and phylogeny of Bridgeoporus nobilissimus have been analyzed. DNA was extracted from spores collected from individual fruiting bodies representing six geographically distinct populations in Oregon and Washington. Spore samples collected contained low levels of bacteria, yeast and a filamentous fungal species. Using taxon-specific PCR primers, it was possible to discriminate among rDNA from bacteria, yeast, a filamentous associate and B. nobilissimus. Nuclear rDNA internal transcribed spacer (ITS) region sequences of B. nobilissimus were compared among individuals representing six populations and were found to have less than 2% variation. These sequences also were used to design dual and nested PCR primers for B. nobilissimus-specific amplification. Mitochondrial small-subunit rDNA sequences were used in a phylogenetic analysis that placed B. nobilissimus in the hymenochaetoid clade, where it was associated with Oxyporus and Schizopora.

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tuskan, Gerald A; Gunter, Lee E; DiFazio, Stephen P

The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis -type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequencemore » assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.« less

Genetic diversity based on 28S rDNA sequences among populations of Culex quinquefasciatus collected at different locations in Tamil Nadu, India.

PubMed

Sakthivelkumar, S; Ramaraj, P; Veeramani, V; Janarthanan, S

2015-09-01

The basis of the present study was to distinguish the existence of any genetic variability among populations of Culex quinquefasciatus which would be a valuable tool in the management of mosquito control programmes. In the present study, population of Cx. quinquefasciatus collected at different locations in Tamil Nadu were analyzed for their genetic variation based on 28S rDNA D2 region nucleotide sequences. A high degree of genetic polymorphism was detected in the sequences of D2 region of 28S rDNA on the predicted secondary structures in spite of high nucleotide sequence similarity. The findings based on secondary structure using rDNA sequences suggested the existence of a complex genotypic diversity of Cx. quinquefasciatus population collected at different locations of Tamil Nadu, India. This complexity in genetic diversity in a single mosquito population collected at different locations is considered an important issue towards their influence and nature of vector potential of these mosquitoes.

Evolutionary genomics of miniature inverted-repeat transposable elements (MITEs) in Brassica.

PubMed

Nouroz, Faisal; Noreen, Shumaila; Heslop-Harrison, J S

2015-12-01

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in most eukaryotic genomes. We aimed to characterize various MITEs families in Brassica in terms of their presence, sequence characteristics and evolutionary activity. Dot plot analyses involving comparison of homoeologous bacterial artificial chromosome (BAC) sequences allowed identification of 15 novel families of mobile MITEs. Of which, 5 were Stowaway-like with TA Target Site Duplications (TSDs), 4 Tourist-like with TAA/TTA TSDs, 5 Mutator-like with 9-10 bp TSDs and 1 novel MITE (BoXMITE1) flanked by 3 bp TSDs. Our data suggested that there are about 30,000 MITE-related sequences in Brassica rapa and B. oleracea genomes. In situ hybridization showed one abundant family was dispersed in the A-genome, while another was located near 45S rDNA sites. PCR analysis using primers flanking sequences of MITE elements detected MITE insertion polymorphisms between and within the three Brassica (AA, BB, CC) genomes, with many insertions being specific to single genomes and others showing evidence of more recent evolutionary insertions. Our BAC sequence comparison strategy enables identification of evolutionarily active MITEs with no prior knowledge of MITE sequences. The details of MITE families reported in Brassica enable their identification, characterization and annotation. Insertion polymorphisms of MITEs and their transposition activity indicated important mechanism of genome evolution and diversification. MITE families derived from known Mariner, Harbinger and Mutator DNA transposons were discovered, as well as some novel structures. The identification of Brassica MITEs will have broad applications in Brassica genomics, breeding, hybridization and phylogeny through their use as DNA markers.

An axenic culture system for fruiting body formation by an edible bolete phylogenetically related to culinary-medicinal penny bun mushroom, Boletus edulis Bull.:Fr. strains from China.

PubMed

Fu, Shao Chun; Zhang, Mei Yan; Shang, Xiao Dong; Chen, Ming Jie; Tan, Qi

2011-01-01

The ability of two freshly isolated Boletus stains to fruit under axenic conditions was tested using different solid and liquid nutrient media. One strain (YNCX04) produced numerous primordia from which fruiting bodies, 12 mm and 10 mm in length, with grey, convex pilei, and yellow-white, clavate stipes developed between 15 and 30 d after inoculation of fungal mycelium onto a solid medium consisting of mineral salts, thiamine, glucose, potato, an extract of Cunninghamia lanceolata root, and agar. The other strain (YNB200) produced numerous primordia but no sporophores. Strain YNCX04 lost the ability to form fruiting bodies in axenic culture 6 mo after initial isolation but retained the ability to form primordia for up to 18 mo. Based on internal transcribed spacer sequencing data, strains YNB200 and YNCX04 formed a sub-cluster together with four previously designated Boletus edulis strains from China. Phylogenetic analysis placed the Chinese strains closer to B. aestivalis than to European and North American strains of B. edulis, although a 29-bp fragment specific to all the B. aestivalis strains was absent from all the Chinese strains. Furthermore, partial 18S rDNA sequences from strains YNB200 and YNCX04 exhibited 98% similarity with an 18S rDNA sequence from B. edulis strain Be3. Further molecular studies are indicated to more accurately establish the taxonomic positions ofF3 and F4-3, as well as the Chinese strains designated as B. edulis.

PHYLOGENETIC RELATIONSHIP OF ALEXANDRIUM MONILATUM (DINOPHYCEAE) TO OTHER ALEXANDRIUM SPECIES BASED ON 18S RIBOSOMAL RNA GENE SEQUENCES

EPA Science Inventory

The phylogenetic relationship of Alexandrium monilatum to other Alexandrium spp. was explored using 18S rDNA sequences. Maximum likelilhood phylogenetic analysis of the combined rDNA sequences established that A. monilatum paired with Alexandrium taylori and that the pair was the...

PHYLOGENETIC RELATIONSHIP OF ALEXANDRIUM MONILATUM (DINOPHYCAE)TO OTHER ALEXANDRIUM SPECIES BASED ON 18S RIBOSOMAL RNA GENE SEQUENCES

EPA Science Inventory

The phylogenetic relationship of Alexandrium monilatum to other Alexandrium spp. was explored using 18S rDNA sequences. Maximum likelihood phylogenetic analysis of the combined rDNA sequences established that A. monilatum paired with Alexandrium taylori and that the pair was the ...

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

PubMed

Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

2008-10-01

In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However, studies on the accuracy of the software packages have given highly varied results, and interpretation of results remains difficult for most technicians, and even for clinical microbiologists. To fully utilize 16S rDNA sequencing in clinical microbiology, better guidelines are needed for interpretation of the identification results, and additional/supplementary methods are necessary for bacterial species that cannot be identified confidently by 16S rDNA sequencing alone.

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

PubMed

Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

2016-12-01

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

Diagnostics of Neisseriaceae and Moraxellaceae by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms

PubMed Central

Harmsen, Dag; Singer, Christian; Rothgänger, Jörg; Tønjum, Tone; Sybren de Hoog, Gerrit; Shah, Haroun; Albert, Jürgen; Frosch, Matthias

2001-01-01

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes. PMID:11230407

Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

PubMed

Mahelka, Václav; Kopecky, David; Baum, Bernard R

2013-09-01

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture.

PubMed

Wagner, K; Springer, B; Pires, V P; Keller, P M

2018-05-03

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Molecular characterization of kudoid parasites (Myxozoa: Multivalvulida) from somatic muscles of Pacific bluefin (Thunnus orientalis) and yellowfin (T. albacores) tuna.

PubMed

Abe, Niichiro; Maehara, Tomofumi

2013-06-01

The public health importance of Kudoa infection in fish remains unclear. Recently in Japan a Kudoa species, K. septempunctata, was newly implicated as a causative agent of unidentified food poisoning related to the consumption of raw olive flounder. Other marine fishery products are also suspected as causative raw foods of unidentified food poisoning. For this study, we detected kudoid parasites from sliced raw muscle tissues of a young Pacific bluefin and an adult yellowfin tuna. No cyst or pseudocyst was evident in muscles macroscopically, but pseudocysts were detected in both samples histologically. One substitution (within 1100 bp overlap) and ten substitutions (within 753 bp overlap) were found respectively between the partial sequences of 18S and 28S rDNAs from both isolates. Nucleotide sequence similarity searching of 18S and 28S rDNAs from both isolates showed the highest identity with those of K. neothunni from tuna. Based on the spore morphology, the mode of parasitism, and the nucleotide sequence similarity, these isolates from a Pacific bluefin and a yellowfin tuna were identified as K. neothunni. Phylogenetic analysis of the 28S rDNA sequence revealed that K. neothunni is classifiable into two genotypes: one from Pacific bluefin and the other from yellowfin tuna. Recently, an unidentified kudoid parasite morphologically and genetically similar K. neothunni were detected from stocked tuna samples in unidentified food poisoning cases in Japan. The possibility exists that K. neothunni, especially from the Pacific bluefin tuna, causes food poisoning, as does K. septempunctata.

[Identification and phylogenetic analysis of one strain of Lactobacillus delbrueckii subsp. bulgaricus separated from yoghourt].

PubMed

Wang, Chuan; Zhang, Chaowu; Pei, Xiaofang; Liu, Hengchuan

2007-11-01

For being further applied and studied, one strain of Lactobacillus delbrueckii subsp. bulgaricus (wch9901) separated from yoghourt which had been identified by phenotype characteristic analysis was identified by 16S rDNA and phylogenetic analyzed. The 16S rDNA of wch9901 was amplified with the genomic DNA of wch9901 as template, and the conservative sequences of the 16S rDNA as primers. Inserted 16S rDNA amplified into clonal vector pGEM-T under the function of T4 DNA ligase to construct recombined plasmid pGEM-wch9901 16S rDNA. The recombined plasmid was identified by restriction enzyme digestion, and the eligible plasmid was presented to sequencing company for DNA sequencing. Nucleic acid sequence was blast in GenBank and phylogenetic tree was constructed using neighbor-joining method of distance methods by Mega3.1 soft. Results of blastn showed that the homology of 16S rDNA of wch9901 with the 16S rDNA of Lactobacillus delbrueckii subsp. bulgaricus strains was higher than 96%. On the phylogenetic tree, wch9901 formed a separate branch and located between Lactobacillus delbrueckii subsp. bulgaricus LGM2 evolution branch and another evolution branch which was composed of Lactobacillus delbrueckii subsp. bulgaricus DL2 evolution cluster and Lactobacillus delbrueckii subsp. bulgaricus JSQ evolution cluster. The distance between wch9901 evolution branch and Lactobacillus delbrueckii subsp. bulgaricus LGM2 evolution branch was the closest. wch9901 belonged to Lactobacillus delbrueckii subsp. bulgaricus. wch9901 showed the closest evolution relationship to Lactobacillus delbrueckii subsp. bulgaricus LGM2.

Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India

PubMed Central

Peddayelachagiri, Bhavani V.; Paul, Soumya; Nagaraj, Sowmya; Gogoi, Madhurjya; Sripathy, Murali H.; Batra, Harsh V.

2016-01-01

Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha based PCR assay when evaluated on the Burkholderia isolates of this study, it was found to be highly specific (100%) in its detection feature and a clear detection sensitivity of 10 pg/μl of purified gDNA was recorded. Nucleotide sequence variations of bdha among interspecies, as per in silico analysis, ranged from 8 to 29% within the target stretch of 730 bp highlighting the potential utility of bdha sequencing method in specific detection of Burkholderia species. Further, sequencing of the 730 bp bdha PCR amplicon of each Burkholderia strain isolated could differentiate the species and the data was comparable with recA sequence data of the strains. All sequencing results obtained were submitted to NCBI database. Bayesian phylogenetic analysis of bdha in comparison with recA and 16S rDNA showed that the bdha gene provided comparable identification of Burkholderia species. PMID:27632353

The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism.

PubMed

Schwelm, Arne; Berney, Cédric; Dixelius, Christina; Bass, David; Neuhauser, Sigrid

2016-12-01

Clubroot disease caused by Plasmodiophora brassicae is one of the most important diseases of cultivated brassicas. P. brassicae occurs in pathotypes which differ in the aggressiveness towards their Brassica host plants. To date no DNA based method to distinguish these pathotypes has been described. In 2011 polymorphism within the 28S rDNA of P. brassicae was reported which potentially could allow to distinguish pathotypes without the need of time-consuming bioassays. However, isolates of P. brassicae from around the world analysed in this study do not show polymorphism in their LSU rDNA sequences. The previously described polymorphism most likely derived from soil inhabiting Cercozoa more specifically Neoheteromita-like glissomonads. Here we correct the LSU rDNA sequence of P. brassicae. By using FISH we demonstrate that our newly generated sequence belongs to the causal agent of clubroot disease. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Ribosomal DNA sequence heterogeneity reflects intraspecies phylogenies and predicts genome structure in two contrasting yeast species.

PubMed

West, Claire; James, Stephen A; Davey, Robert P; Dicks, Jo; Roberts, Ian N

2014-07-01

The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of closely related organisms, and discuss how it could be extended to future studies of multilocus rDNA systems. [concerted evolution; genome hydridisation; phylogenetic analysis; ribosomal DNA; whole genome sequencing; yeast]. © The Author(s) 2014. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.

Armillaria phylogeny based on tef-1α sequences suggests ongoing divergent speciation within the boreal floristic kingdom

Treesearch

Ned B. Klopfenstein; John W. Hanna; Amy L. Ross-Davis; Jane E. Stewart; Yuko Ota; Rosario Medel-Ortiz; Miguel Armando Lopez-Ramirez; Ruben Damian Elias-Roman; Dionicio Alvarado-Rosales; Mee-Sook Kim

2013-01-01

Armillaria plays diverse ecological roles in forests worldwide, which has inspired interest in understanding phylogenetic relationships within and among species of this genus. Previous rDNA sequence-based phylogenetic analyses of Armillaria have shown general relationships among widely divergent taxa, but rDNA sequences were not reliable for separating closely related...

Correlation of 16S Ribosomal DNA Signature Sequences with Temperature-Dependent Growth Rates of Mesophilic and Psychrotolerant Strains of the Bacillus cereus Group

PubMed Central

Prüß, Birgit M.; Francis, Kevin P.; von Stetten, Felix; Scherer, Siegfried

1999-01-01

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance. PMID:10198030

[Molecular identification of medicinal plant genus Uncaria in Guizhou].

PubMed

Gang, Tao; Liu, Tao; Zhu, Ying; Liu, Zuo-Yi

2008-06-01

To analyze rDNA ITS regions of the Medicinal Plant Genus Uncaria in Guizhou and construct their phylogenetic tree in order to supply molecular evidence of taxonomy and identification of these Medicinal Plants in genetic level. The ITS gene fragments of the 4 Medicinal Plants were PCR amplified and sequenced. The rDNA ITS regions were analyzed by means of the software of ClustalX, BioEdit and PAUP* 4.0 beta 10. The entire sequences of rDNA ITS1, ITS2, and 5.8S rDNA were obtained, The Maximum-parsimony tree of four ITS regions together with those of similar sequences from GenBank were found, as Mitrayna rubrostipulata (AJ492621 ) and Mitragyna rubrostipulata (AJ605988) were designated as outgroup. The 4 medicinal plants are the 4 species in the genus Uncaria, and are mostly similar to the Uncaria rhynhcophylla.

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

PubMed Central

Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

1980-01-01

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

Identification of a new myxosporean parasite Thelohanellus indiana n. sp. (Myxosporea: Myxobolidae) isolated from three major organs of goldfish, Carassius auratus L. highlighted with its morphological and SSU rDNA sequence based molecular description.

PubMed

Saha, Mandira; Bandyopadhyay, Probir Kumar

2018-05-24

Fish mortality and poor growth in surviving fish contribute substantial losses to the ornamental fish farms of India and revealed an infection of a new myxosporidian parasite Thelohanellus indiana n. sp. which has become one of the most important limiting factors for successful aquaculture management. The parasite infects Carassius auratus, an Indian goldfish, described on the basis of myxospores morphology and amplification of a part of 18 S rDNA gene. Three major attaching site of fish body have been explored for showing the location of attachment for the parasites. The whitish cysts of the parasites are about 2.5-3.5 mm contains large amount of lemon shaped mature myxospores measuring 12.1-15.2 (13.8) × 7.5-8.8 (8) μm. A single round or elliptical polar capsule located only at the anterior pole of the spore having 6.2-7.2 (6.8) × 3.3-4.7 (4.0) μm in diameter. The morphological characters have been assessed by both the light and scanning electron microscope. The most differentiating feature from closely related species was carried out by morpho-taxonomic affinities with previously described species which are tremendously supported by molecular taxonomy by partial sequencing of the 18 S rDNA gene resulted in a total of 2101 bp fragment of newly obtained SSU rRNA gene sequence of the new species which exhibit 79-91% homogeneity with other closely related species available in GenBank. The BLAST search of Thelohanellus sp. did not matches with any available sequences in GenBank and the phylogenetic analysis revealed that the novel species were sister to T. habibpuri and T. caudatus, in the Thelohanellus clade and form a closest neighboring branch as a subclade in phylogenetic tree from which the new Thelohanellus parasite is being placed. Both the branches are originating from monophyletic clade that are strongly supported by bootstrap values which indicate clearly about independent position of T. indiana n. sp. Copyright © 2018. Published by Elsevier Ltd.

Molecular identification of environmental bacteria in indoor air in the domestic home: description of a new species of Exiguobacterium.

PubMed

Yuan, Ivan; Xu, Jiru; Millar, B Cherie; Dooley, James S G; Rooney, Paul J; Alexander, H Denis; Moore, John E

2007-02-01

The quality of indoor air in terms of its bioaerosol composition with microorganisms is important due to its potential aetiological role in development of conditions such as Sick Building Syndrome. Hence, laboratory identification of bacteriological components in any bioaerosol from buildings may help elucidate the role of such organisms in disease states, particularly allergy-related conditions. A molecular method was developed employing universal or "broad-range" eubacterial PCR to help identify environmental culturable bacteria from domestic household air. In a "proof of concept" experiment, 16S rDNA PCR was performed on a collection of bacterial isolates originating from indoor air in the domestic home. 16S rDNA PCR was performed using a set of universal primers to successfully generate an amplicon of approximately 1400 bp, which was sequenced to obtain each isolate's identity. Sequence analysis was able to identify 12/13 of the isolates, whereby the majority were Gram-positive (12/13). Nine different genera were identified from the 13 isolates examined, of which, 12/13 were Gram-positive, with the exception being Moraxella osloensis, which was Gram-negative, as well as a novel species of Exiguobacterium. The closest phylogenetic neighbour of the wildtype isolate to a named species within this genus was E. aestuarii (1364/1384 bases; 98.4% homology), followed by E. marinum (97.5%) and with E. acetylicum being the most distantly related of all the described species. On account of this divergence within the 16S rDNA gene operon of the unknown Exiguobacterium isolate, we believe this isolate to represent a novel species of Exiguobacterium, which we have tentatively named Exiguobacterium belfastensis. Although from this study, these organisms are usually unlikely to be clinically significant to healthy individuals with a competent immune system, we recommend that molecular identification methods are used, if considered necessary, as an adjunct to first line phenotypic identification schemes, where a definitive identification is required. When the use of molecular identification methods is justified, employment of partial 16S rDNA PCR and sequencing provides a valuable and reliable method of identification of environmental bacteria in the home. This study demonstrates the usefulness of such methods and a full and comprehensive study is now required to examine the diversity of bacteria in indoor air in the home, with particular emphasis on the risk of such environmental organisms to immunosurpressed patients, such as those with haematological malignancies and who are neutropenic.

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

PubMed

van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

1991-06-01

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores.

PubMed

Atibalentja, N; Noel, G R; Ciancio, A

2004-03-01

For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 x 10 endospores ml(-1) were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis.

Paecilomyces niveus Stolk & Samson, 1971 (Ascomycota: Thermoascaceae) as a pathogen of Nasonovia ribisnigri (Mosley, 1841) (Hemiptera, Aphididae) in Brazil.

PubMed

Zawadneak, M A C; Pimentel, I C; Robl, D; Dalzoto, P; Vicente, V; Sosa-Gómez, D R; Porsani, M; Cuquel, F L

2015-11-01

Nasonovia ribisnigri is a key pest of lettuce (Lactuca sativa L.) in Brazil that requires alternative control methods to synthetic pesticides. We report, for the first time, the occurrence of Paecilomyces niveus as an entomopathogen of the aphid Nasonovia ribisnigri in Pinhais, Paraná, Brazil. Samples of mummified aphids were collected from lettuce crops. The fungus P. niveus (PaePR) was isolated from the insect bodies and identified by macro and micromorphology. The species was confirmed by sequencing Internal Transcribed Spacer (ITS) rDNA. We obtained a sequence of 528 bp (accession number HQ441751), which aligned with Byssochlamys nivea strains (100% identities). In a bioassay, 120 h after inoculation of N. ribisnigri with pathogenic P. niveus had an average mortality of 74%. The presence of P. niveus as a natural pathogen of N. ribisnigri in Brazil suggests that it may be possible to employ P. niveus to minimize the use of chemical insecticides.

Ribosomal DNA intergenic spacer sequence in foxtail millet, Setaria italica (L.) P. Beauv. and its characterization and application to typing of foxtail millet landraces.

PubMed

Fukunaga, Kenji; Ichitani, Katsuyuki; Taura, Satoru; Sato, Muneharu; Kawase, Makoto

2005-02-01

We determined the sequence of ribosomal DNA (rDNA) intergenic spacer (IGS) of foxtail millet isolated in our previous study, and identified subrepeats in the polymorphic region. We also developed a PCR-based method for identifying rDNA types based on sequence information and assessed 153 accessions of foxtail millet. Results were congruent with our previous works. This study provides new findings regarding the geographical distribution of rDNA variants. This new method facilitates analyses of numerous foxtail millet accessions. It is helpful for typing of foxtail millet germplasms and elucidating the evolution of this millet.

Genetic diversity of Burkholderia (Proteobacteria) species from the Caatinga and Atlantic rainforest biomes in Bahia, Brazil.

PubMed

Santini, A C; Santos, H R M; Gross, E; Corrêa, R X

2013-03-11

The genus Burkholderia (β-Proteobacteria) currently comprises more than 60 species, including parasites, symbionts and free-living organisms. Several new species of Burkholderia have recently been described showing a great diversity of phenotypes. We examined the diversity of Burkholderia spp in environmental samples collected from Caatinga and Atlantic rainforest biomes of Bahia, Brazil. Legume nodules were collected from five locations, and 16S rDNA and recA genes of the isolated microorganisms were analyzed. Thirty-three contigs of 16S rRNA genes and four contigs of the recA gene related to the genus Burkholderia were obtained. The genetic dissimilarity of the strains ranged from 0 to 2.5% based on 16S rDNA analysis, indicating two main branches: one distinct branch of the dendrogram for the B. cepacia complex and another branch that rendered three major groups, partially reflecting host plants and locations. A dendrogram designed with sequences of this research and those designed with sequences of Burkholderia-type strains and the first hit BLAST had similar topologies. A dendrogram similar to that constructed by analysis of 16S rDNA was obtained using sequences of the fragment of the recA gene. The 16S rDNA sequences enabled sufficient identification of relevant similarities and groupings amongst isolates and the sequences that we obtained. Only 6 of the 33 isolates analyzed via 16S rDNA sequencing showed high similarity with the B. cepacia complex. Thus, over 3/4 of the isolates have potential for biotechnological applications.

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

PubMed

Zhu, X Q; Gasser, R B

1998-06-01

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

A global meta-analysis of Tuber ITS rDNA sequences: species diversity, host associations and long-distance dispersal

Treesearch

Gregory M. Bonito; Andrii P. Gryganskyi; James M. Trappe; Rytas Vilgalys

2010-01-01

Truffles (Tuber) are ectomycorrhizal fungi characterized by hypogeous fruitbodies. Their biodiversity, host associations and geographical distributions are not well documented. ITS rDNA sequences of Tuber are commonly recovered from molecular surveys of fungal communities, but most remain insufficiently identified making it...

BIOCHEMICAL AND PHYLOGENETIC CHARACTERIZATION OF TWO NOVEL DEEP-SEA THERMOCOCCUS ISOLATES WITH POTENTIALLY BIOTECHNOLOGICAL APPLICATIONS

EPA Science Inventory

The partial 16S rDNA gene sequences of two thermophilic archaeal strains, TY and TYS, previously isolated from the Guaymas Basin hydrothermal vent site were determined. Lipid analyses and a comparative analysis performed with 16S rDNA sequences of similar thermophilic species sho...

Molecular and phylogenetic characterizations of an Eimeria krijgsmanni Yakimoff & Gouseff, 1938 (Apicomplexa: Eimeriidae) mouse intestinal protozoan parasite by partial 18S ribosomal RNA gene sequence analysis.

PubMed

Takeo, Toshinori; Tanaka, Tetsuya; Matsubayashi, Makoto; Maeda, Hiroki; Kusakisako, Kodai; Matsui, Toshihiro; Mochizuki, Masami; Matsuo, Tomohide

2014-08-01

Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA-intercalating agent.

PubMed

Zhu, X Q; Chilton, N B; Gasser, R B

1998-05-01

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold-supplemented gels. The latter was achieved because of differences (approximately 0.1-8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11-39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR-amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans.

PubMed

Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

2013-11-01

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12,766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

PubMed Central

Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

2013-01-01

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades. PMID:23838690

Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

PubMed

Tian, Yang; Li, Yan Hong

2017-01-01

To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814).

PubMed

Śliwińska-Jewsiewicka, A; Kuciński, M; Kirtiklis, L; Dobosz, S; Ocalewicz, K; Jankun, Malgorzata

2015-08-01

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3-6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii)

PubMed Central

Klosterman, Steven J.; Anchieta, Amy; McRoberts, Neil; Koike, Steven T.; Subbarao, Krishna V.; Voglmayr, Hermann; Choi, Young-Joon; Thines, Marco; Martin, Frank N.

2016-01-01

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management. PMID:24964150

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

PubMed

Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

2005-10-01

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

Preferential cleavage sites for Sau3A restriction endonuclease in human ribosomal DNA.

PubMed

Kupriyanova, N S; Kirilenko, P M; Netchvolodov, K K; Ryskov, A P

2000-07-21

Previous studies of cloned ribosomal DNA (rDNA) variants isolated from the cosmid library of human chromosome 13 have revealed some disproportion in representativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvolodov, P. M. Kirilenko, B. I. Kapanadze, N. K. Yankovsky, and A. P. Ryskov, Mol. Biol. 30, 51-60, 1996). Here we show nonrandom cleavage of human rDNA with Sau3A or its isoshizomer MboI under mild hydrolysis conditions. The hypersensitive cleavage sites were found to be located in the ribosomal intergenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb upstream of the rRNA transcription start point. This finding is based on sequencing mapping of the rDNA insert ends in randomly selected cosmid clones of human chromosome 13 and on the data of digestion kinetics of cloned and noncloned human genomic rDNA with Sau3A and MboI. The results show that a methylation status and superhelicity state of the rIGS have no effect on cleavage site sensitivity. It is interesting that all primary cleavage sites are adjacent to or entering into Alu or Psi cdc 27 retroposons of the rIGS suggesting a possible role of neighboring sequences in nuclease accessibility. The results explain nonequal representation of rDNA sequences in the human genomic DNA library used for this study. Copyright 2000 Academic Press.

Barcode Identifiers as a Practical Tool for Reliable Species Assignment of Medically Important Black Yeast Species

PubMed Central

Heinrichs, Guido; de Hoog, G. Sybren

2012-01-01

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Starting from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives. PMID:22785187

Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

PubMed

Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

2004-06-15

The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.

Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans.

PubMed

Nakano, Tadao; Okamoto, Munehiro; Ikeda, Yatsukaho; Hasegawa, Hideo

2006-12-01

Sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, nuclear internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA), and 5S rDNA of Enterobius vermicularis from captive chimpanzees in five zoos/institutions in Japan were analyzed and compared with those of pinworm eggs from humans in Japan. Three major types of variants appearing in both CO1 and ITS2 sequences, but showing no apparent connection, were observed among materials collected from the chimpanzees. Each one of them was also observed in pinworms in humans. Sequences of 5S rDNA were identical in the materials from chimpanzees and humans. Phylogenetic analysis of CO1 gene revealed three clusters with high bootstrap value, suggesting considerable divergence, presumably correlated with human evolution, has occurred in the human pinworms. The synonymy of E. gregorii with E. vermicularis is supported by the molecular evidence.

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

PubMed

Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

2016-04-15

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Novel division level bacterial diversity in a Yellowstone hot spring.

PubMed

Hugenholtz, P; Pitulle, C; Hershberger, K L; Pace, N R

1998-01-01

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID:25653642

Karyotype divergence and spreading of 5S rDNA sequences between genomes of two species: darter and emerald gobies ( Ctenogobius , Gobiidae).

PubMed

Lima-Filho, P A; Bertollo, L A C; Cioffi, M B; Costa, G W W F; Molina, W F

2014-01-01

Karyotype analyses of the cryptobenthic marine species Ctenogobius boleosoma and C. smaragdus were performed by means of classical and molecular cytogenetics, including physical mapping of the multigene 18S and 5S rDNA families. C. boleosoma has 2n = 44 chromosomes (2 submetacentrics + 42 acrocentrics; FN = 46) with a single chromosome pair each carrying 18S and 5S ribosomal sites; whereas C. smaragdus has 2n = 48 chromosomes (2 submetacentrics + 46 acrocentrics; FN = 50), also with a single pair bearing 18S rDNA, but an extensive increase in the number of GC-rich 5S rDNA sites in 21 chromosome pairs. The highly divergent karyotypes among Ctenogobius species contrast with observations in several other marine fish groups, demonstrating an accelerated rate of chromosomal evolution mediated by both chromosomal rearrangements and the extensive dispersion of 5S rDNA sequences in the genome. © 2014 S. Karger AG, Basel.

Coinfection by Ureaplasma spp., Photobacterium damselae and an Actinomyces-like microorganism in a bottlenose dolphin (Tursiops truncatus) with pleuropneumonia stranded along the Adriatic coast of Italy.

PubMed

Di Francesco, Gabriella; Cammà, Cesare; Curini, Valentina; Mazzariol, Sandro; Proietto, Umberto; Di Francesco, Cristina Esmeralda; Ferri, Nicola; Di Provvido, Andrea; Di Guardo, Giovanni

2016-04-01

A case of pleuropneumonia is reported in an adult male bottlenose dolphin (Tursiops truncatus) found stranded in 2014 along the Central Adriatic coast of Italy. A severe pyogranulomatous pneumonia and thoracic lymphadenopathy were present at necropsy. Numerous Splendore-Hoeppli bodies were found microscopically scattered throughout the lung. Histochemical evidence of Actinomyces-like organisms was obtained from the pulmonary parenchyma, with a strain of Photobacterium damselae subsp. piscicida and Ureaplasma spp. being also isolated from the same tissue. For the latter, a genome fragment of approximately 1400 bp from the 16s rDNA was amplified and sequenced. BLAST analysis revealed 100% identity with an uncultured Ureaplasma spp. (JQ193826.1). Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

PubMed

Lawton, Samantha J; Weis, Allison M; Byrne, Barbara A; Fritz, Heather; Taff, Conor C; Townsend, Andrea K; Weimer, Bart C; Mete, Aslı; Wheeler, Sarah; Boyce, Walter M

2018-05-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

Intragenomic sequence variation at the ITS1 - ITS2 region and at the 18S and 28S nuclear ribosomal DNA genes of the New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae: mollusca)

USGS Publications Warehouse

Hoy, Marshal S.; Rodriguez, Rusty J.

2013-01-01

Molecular genetic analysis was conducted on two populations of the invasive non-native New Zealand mud snail (Potamopyrgus antipodarum), one from a freshwater ecosystem in Devil's Lake (Oregon, USA) and the other from an ecosystem of higher salinity in the Columbia River estuary (Hammond Harbor, Oregon, USA). To elucidate potential genetic differences between the two populations, three segments of nuclear ribosomal DNA (rDNA), the ITS1-ITS2 regions and the 18S and 28S rDNA genes were cloned and sequenced. Variant sequences within each individual were found in all three rDNA segments. Folding models were utilized for secondary structure analysis and results indicated that there were many sequences which contained structure-altering polymorphisms, which suggests they could be nonfunctional pseudogenes. In addition, analysis of molecular variance (AMOVA) was used for hierarchical analysis of genetic variance to estimate variation within and among populations and within individuals. AMOVA revealed significant variation in the ITS region between the populations and among clones within individuals, while in the 5.8S rDNA significant variation was revealed among individuals within the two populations. High levels of intragenomic variation were found in the ITS regions, which are known to be highly variable in many organisms. More interestingly, intragenomic variation was also found in the 18S and 28S rDNA, which has rarely been observed in animals and is so far unreported in Mollusca. We postulate that in these P. antipodarum populations the effects of concerted evolution are diminished due to the fact that not all of the rDNA genes in their polyploid genome should be essential for sustaining cellular function. This could lead to a lessening of selection pressures, allowing mutations to accumulate in some copies, changing them into variant sequences.                   

Isolation, evaluation and characterization of Bacillus subtilis from cotton rhizospheric soil with biocontrol activity against Fusarium oxysporum.

PubMed

Gajbhiye, Archana; Rai, Alok R; Meshram, Sudhir U; Dongre, A B

2010-07-01

Present investigation is based on the isolation of Bacillus subtilis from cotton rhizosphere and their evaluation as biocontrol agent against Fusarium oxysporum. The production of extracellular hydrolytic enzyme was studied for determining the antagonism. 43% of 21 isolates were identified under the B. subtilis group on the basis of biochemical characterization. 38% isolates showed competitive activity against Fusarium oxysporum and exhibit more than 50% mycelial inhibition in dual culture bioassay. The pot assay of cotton by seed treatment and soil amendment technique under green house condition showed the competent activity of the isolates in preventing the wilting of cotton seedlings due to F. oxysporum infection. SVI values of 30 day old seedlings indicated that the soil inoculation with B. subtilis BP-2 and seed treatment with B. subtilis BP-9 significantly promoted the growth of cotton seedlings. RAPD profiling revealed the diversity in the Bacillus subtilis group, ranging from 10 to 32%. The discriminative pattern among the isolates belonging to the same species was validated by 16S rDNA partial sequencing which identified them into four different strains of B. subtilis.

Multiple horizontal transfers of nuclear ribosomal genes between phylogenetically distinct grass lineages.

PubMed

Mahelka, Václav; Krak, Karol; Kopecký, David; Fehrer, Judith; Šafář, Jan; Bartoš, Jan; Hobza, Roman; Blavet, Nicolas; Blattner, Frank R

2017-02-14

The movement of nuclear DNA from one vascular plant species to another in the absence of fertilization is thought to be rare. Here, nonnative rRNA gene [ribosomal DNA (rDNA)] copies were identified in a set of 16 diploid barley ( Hordeum ) species; their origin was traceable via their internal transcribed spacer (ITS) sequence to five distinct Panicoideae genera, a lineage that split from the Pooideae about 60 Mya. Phylogenetic, cytogenetic, and genomic analyses implied that the nonnative sequences were acquired between 1 and 5 Mya after a series of multiple events, with the result that some current Hordeum sp. individuals harbor up to five different panicoid rDNA units in addition to the native Hordeum rDNA copies. There was no evidence that any of the nonnative rDNA units were transcribed; some showed indications of having been silenced via pseudogenization. A single copy of a Panicum sp. rDNA unit present in H. bogdanii had been interrupted by a native transposable element and was surrounded by about 70 kbp of mostly noncoding sequence of panicoid origin. The data suggest that horizontal gene transfer between vascular plants is not a rare event, that it is not necessarily restricted to one or a few genes only, and that it can be selectively neutral.

Evolutionary Dynamics of 5S rDNA and Recurrent Association of Transposable Elements in Electric Fish of the Family Gymnotidae (Gymnotiformes): The Case of Gymnotus mamiraua.

PubMed

da Silva, Maelin; Barbosa, Patricia; Artoni, Roberto F; Feldberg, Eliana

2016-01-01

Gymnotidae is a family of electric fish endemic to the Neotropics consisting of 2 genera: Electrophorus and Gymnotus. The genus Gymnotus is widely distributed and is found in all of the major Brazilian river systems. Physical and molecular mapping data for the ribosomal DNA (rDNA) in this genus are still scarce, with its chromosomal location known in only 11 species. As other species of Gymnotus with 2n = 54 chromosomes from the Paraná-Paraguay basin, G. mamiraua was found to have a large number of 5S rDNA sites. Isolation and cloning of the 5S rDNA sequences from G. mamiraua identified a fragment of a transposable element similar to the Tc1/mariner transposon associated with a non-transcribed spacer. Double fluorescence in situ hybridization analysis of this element and the 5S rDNA showed that they were colocalized on several chromosomes, in addition to acting as nonsyntenic markers on others. Our data show the association between these sequences and suggest that the Tc1 retrotransposon may be the agent that drives the spread of these 5S rDNA-like sequences in the G. mamiraua genome. © 2016 S. Karger AG, Basel.

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae)

PubMed Central

2012-01-01

Background For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Results Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Conclusions Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others. PMID:22433220

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae).

PubMed

Rodrigues, Débora Silva; Rivera, Miryan; Lourenço, Luciana Bolsoni

2012-03-20

For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others.

Sharp switches between regular and swinger mitochondrial replication: 16S rDNA systematically exchanging nucleotides A<->T+C<->G in the mitogenome of Kamimuria wangi.

PubMed

Seligmann, Hervé

2016-07-01

Swinger DNAs are sequences whose homology with known sequences is detected only by assuming systematic exchanges between nucleotides. Nine symmetric (X<->Y, i.e. A<->C) and fourteen asymmetric (X->Y->Z, i.e. A->C->G) exchanges exist. All swinger DNA previously detected in GenBank follow the A<->T+C<->G exchange, while mitochondrial swinger RNAs distribute among different swinger types. Here different alignment criteria detect 87 additional swinger mitochondrial DNAs (86 from insects), including the first swinger gene embedded within a complete genome, corresponding to the mitochondrial 16S rDNA of the stonefly Kamimuria wangi. Other Kamimuria mt genome regions are "regular", stressing unanswered questions on (a) swinger polymerization regulation; (b) swinger 16S rDNA functions; and (c) specificity to rDNA, in particular 16S rDNA. Sharp switches between regular and swinger replication, together with previous observations on swinger transcription, suggest that swinger replication might be due to a switch in polymerization mode of regular polymerases and the possibility of swinger-encoded information, predicted in primordial genes such as rDNA.

Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

PubMed Central

Guerrini, A M; Ascenzioni, F; Tribioli, C; Donini, P

1985-01-01

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids. Images Fig. 1. Fig. 2. Fig. 4. PMID:3896773

[Sequencing and analysis of the complete mitochondrial genome of the King Cobra, Ophiophagus hannah (Serpents: Elapidae)].

PubMed

Chen, Nian; Lai, Xiao-Ping

2010-07-01

We obtained the complete mitochondrial genome of King Cobra(GenBank accession number: EU_921899) by Ex Taq-PCR, TA-cloning and primer-walking methods. This genome is very similar to other vertebrate, which is 17 267 bp in length and encodes 38 genes (including 13 protein-coding, 2 ribosomal RNA and 23 transfer RNA genes) and two long non-coding regions. The duplication of tRNA-Ile gene forms a new mitochondrial gene rearrangement model. Eight tRNA genes and one protein genes were transcribed from L strand, and the other genes were transcribed genes from H strand. Genes on the H strand show a fairly similar content of Adenosine and Thymine respectively, whereas those on the L strand have higher proportion of A than T. Combined rDNA sequence data (12S+16S rRNA) were used to reconstruct the phylogeny of 21 snake species for which complete mitochondrial genome sequences were available in the public databases. This large data set and an appropriate range of outgroup taxa demonstrated that Elapidae is more closely related to colubridae than viperidae, which supports the traditional viewpoints.

Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout

USGS Publications Warehouse

Rasmussen, C.; Purcell, M.K.; Gregg, J.L.; LaPatra, S.E.; Winton, J.R.; Hershberger, P.K.

2010-01-01

The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of ~740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

Replication and meiotic transmission of yeast ribosomal RNA genes.

PubMed

Brewer, B J; Zakian, V A; Fangman, W L

1980-11-01

The yeast Saccharomyces cerevisiae has approximately 120 genes for the ribosomal RNAs (rDNA) which are organized in tandem within chromosomal DNA. These multiple-copy genes are homogeneous in sequence but can undergo changes in copy number and topology. To determine if these changes reflect unusual features of rDNA metabolism, we have examined both the replication of rDNA in the mitotic cell cycle and the inheritance of rDNA during meiosis. The results indicate that rDNA behaves identically to chromosomal DNA: each rDNA unit is replicated once during the S phase of each cell cycle and each unit is conserved through meiosis. Therefore, the flexibility in copy number and topology of rDNA does not arise from the selective replication of units in each S phase nor by the selective inheritance of units in meiosis.

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

PubMed Central

Burke, W D; Calalang, C C; Eickbush, T H

1987-01-01

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover. Images PMID:2439905

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

PubMed

Han, Jun-ge; Wang, Cheng-bao; Li, Xing-biao; Fan, Yan-yan; Feng, Xiang-ping

2013-10-01

To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

'Candidatus pasteuria usgae' sp. nov., an obligate endoparasite of the phytoparasitic nematode Belonolaimus longicaudatus.

PubMed

Giblin-Davis, R M; Williams, D S; Bekal, S; Dickson, D W; Brito, J A; Becker, J O; Preston, J F

2003-01-01

Taxonomically relevant characteristics of a fastidiously Gram-positive, obligately endoparasitic prokaryote (strain S-1) that uses the phytoparasitic sting nematode Belonolaimus longicaudatus as its host are reviewed. 16S rDNA sequence similarity (> or = 93%) confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans and corroborates morphological, morphometric and host range evidence suggesting a novel taxon. The 16S rDNA sequence of strain S-1 has greatest similarity (96%) to the 16S rDNA sequences of both Pasteuria penetrans from root-knot nematodes (Meloidogyne species) and the recently reported strain of Pasteuria isolated from the soybean cyst nematode Heterodera glycines. Because the obligately endoparasitic nature of prokaryotes in the genus Pasteuria prevents isolation of definitive type strains, strain S-1 is proposed as 'Candidatus Pasteuria usgae' sp. nov.

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

PubMed Central

Hamelin, R C; Bérubé, P; Gignac, M; Bourassa, M

1996-01-01

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated. PMID:8899993

Estimation of the Relative Abundance of Different Bacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

PubMed Central

Wood, Jacqueline; Scott, Karen P.; Avguštin, Gorazd; Newbold, C. James; Flint, Harry J.

1998-01-01

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples. PMID:9758785

Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

PubMed Central

2012-01-01

Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated. Conclusions Counter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection. PMID:22929533

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

PubMed

Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Mu-Chiou; Wang, Li-Tin; Huang, Lina; Lee, Fwu-Ling

2012-10-01

To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing. Copyright © 2012 Society of Chemical Industry.

Use of molecular markers to compare Fusarium verticillioides pathogenic strains isolated from plants and humans.

PubMed

Chang, S C; Macêdo, D P C; Souza-Motta, C M; Oliveira, N T

2013-08-12

Fusarium verticillioides is a pathogen of agriculturally important crops, especially maize. It is considered one of the most important pathogens responsible for fumonisin contamination of food products, which causes severe, chronic, and acute intoxication in humans and animals. Moreover, it is recognized as a cause of localized infections in immunocompetent patients and disseminated infections among severely immunosuppressed patients. Several molecular tools have been used to analyze the intraspecific variability of fungi. The objective of this study was to use molecular markers to compare pathogenic isolates of F. verticillioides and isolates of the same species obtained from clinical samples of patients with Fusarium mycoses. The molecular markers that we used were inter-simple sequence repeat markers (primers GTG5 and GACA4), intron splice site primer (primer EI1), random amplified polymorphic DNA marker (primer OPW-6), and restriction fragment length polymorphism-internal transcribed spacer (ITS) from rDNA. From the data obtained, clusters were generated based on the UPGMA clustering method. The amplification products obtained using primers ITS4 and ITS5 and loci ITS1-5.8-ITS2 of the rDNA yielded fragments of approximately 600 bp for all the isolates. Digestion of the ITS region fragment using restriction enzymes such as EcoRI, DraI, BshI, AluI, HaeIII, HinfI, MspI, and PstI did not permit differentiation among pathogenic and clinical isolates. The inter-simple sequence repeat, intron splice site primer, and random amplified polymorphic DNA markers presented high genetic homogeneity among clinical isolates in contrast to the high variability found among the phytopathogenic isolates of F. verticillioides.

First description of Cryptosporidium parvum in carrier pigeons (Columba livia).

PubMed

Oliveira, Bruno César Miranda; Ferrari, Elis Domingos; da Cruz Panegossi, Mariele Fernanda; Nakamura, Alex Akira; Corbucci, Flávio Sader; Nagata, Walter Bertequini; Dos Santos, Bianca Martins; Gomes, Jancarlo Ferreira; Meireles, Marcelo Vasconcelos; Widmer, Giovanni; Bresciani, Katia Denise Saraiva

2017-08-30

The carrier pigeon and the domestic pigeon are different breeds of the species Columba livia. Carrier pigeons are used for recreational activities such as bird contests and exhibitions. Due to the close contact with humans, these birds may potentially represent a public health risk, since they can host and disseminate zoonotic parasites, such as those belonging to the genus Cryptosporidium (phylum Apicomplexa). The purpose of this work was the detection by microscopic and molecular techniques of Cryptosporidium spp. oocysts in fecal samples of carrier pigeons, and subsequently to sequence the 18S ribosomal RNA marker of positive samples to identify the species. A total of 100 fecal samples were collected individually in two pigeon breeding facilities from Formiga and Araçatuba, cities located in Minas Gerais state and São Paulo state, Brazil, respectively. The age of the birds ranged from one to 12 years; 56 were females and 44 males. Fecal smears were stained with negative malachite green, whereas the molecular characterization was based on the sequence of a ∼800bp fragment of the 18S rRNA gene. Microscopic examination of fecal smears revealed 4% (4/100) oocyst positivity. On the other hand, 7% (7/100) of positivity were found using nested PCR. Three samples were 99% to 100% similar to Cryptosporidium parvum 18S rDNA type A (Genbank AH006572) and the other three samples had 99% to 100% similarity to C. parvum 18S rDNA type B (Genbank AF308600). To our knowledge, this is the first report of C. parvum oocysts in carrier pigeons. Copyright © 2017 Elsevier B.V. All rights reserved.

Intraspecific Genetic Variation and Phylogenetic Analysis of Dirofilaria immitis Samples from Western China Using Complete ND1 and 16S rDNA Gene Sequences

PubMed Central

Liu, Tianyu; Liang, Yinan; Zhong, Xiuqin; Wang, Ning; Hu, Dandan; Zhou, Xuan; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2014-01-01

Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1. PMID:24639299

Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China.

PubMed

Shu, Fan-Fan; Lv, Rui-Qing; Zhang, Yi-Fang; Duan, Gang; Wu, Ding-Yu; Li, Bi-Feng; Yang, Jian-Fa; Zou, Feng-Cai

2012-08-01

On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.

Effects of different management practices on fungal biodiversity in agricultural soils

NASA Astrophysics Data System (ADS)

Borriello, R.; Lumini, E.; Bonfante, P.; Bianciotto, V.

2009-04-01

Symbiotic associations between arbuscular mycorrhizal fungi (AMF) and plant roots are widespread in natural environments and provide a range of benefits to the host plant. These include improved nutrition, enhanced resistance to soil-borne pests, diseases, and drought, as well as tolerance to heavy metals. In addition, the presence of a well developed AMF hyphal network improve the soil structure. As obligate mutualistic symbionts these fungi colonize the roots of many agricultural crops and it is often claimed that agricultural practices (use of fertilizers and biocides, tillage, dominance of monocultures and the growing of non-mycorrhizal crops) are detrimental to AMF. As a result, agro ecosystems impoverished in AMF may not get the fully expected range of benefits from these fungi. Using molecular markers on DNA extracted directly from soil and roots we studied the effects of different management practices (tillage and nitrogen fertilization) on the AMF populations colonizing an experimental agro ecosystem in Central Italy. Fungi in roots and soil were identified by cloning and sequencing a region of ~550bp of the 18S rDNA and ~600bp of the 28S rDNA. In symbiosis with the maize roots we detected only members of Glomeraceae group A that showed decrement in number under nitrogen fertilization. Instead in soil were mainly present members of two AMF groups, respectively Gigasporaceae and Glomeraceae group A. In addition only the low input management practices preserve also members of Diversisporaceae and Glomeraceae group B. From our study we can conclude that agricultural practices can directly or indirectly influence AMF biodiversity. The result of this study highlight the importance and significant effects of the long term nitrogen fertilization and tillage practices on specific groups of fungi playing a key role in arable soils. The research was founded by Biodiversity Project (IPP-CNR) and by SOILSINK (FISR-MIUR)

Molecular genetic diversity of Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914) (Spirurida: Gongylonematidae) from rodents in Southeast Asia.

PubMed

Setsuda, Aogu; Ribas, Alexis; Chaisiri, Kittipong; Morand, Serge; Chou, Monidarin; Malbas, Fidelino; Yunus, Muchammad; Sato, Hiroshi

2018-03-01

More than a dozen Gongylonema spp. (Spirurida: Spiruroidea: Gongylonematidae) have been described from a variety of rodent hosts worldwide. Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914), which dwells in the gastric mucosa of rats such as Rattus norvegicus (Berkenhout) and Rattus rattus (Linnaeus), is currently regarded as a cosmopolitan nematode in accordance with global dispersion of its definitive hosts beyond Asia. To facilitate the reliable specific differentiation of local rodent Gongylonema spp. from the cosmopolitan congener, the genetic characterisation of G. neoplasticum from Asian Rattus spp. in the original endemic area should be considered since the morphological identification of Gongylonema spp. is often difficult due to variations of critical phenotypical characters, e.g. spicule lengths and numbers of caudal papillae. In the present study, morphologically identified G. neoplasticum from 114 rats of seven species from Southeast Asia were selected from archived survey materials from almost 4,500 rodents: Thailand (58 rats), Cambodia (52 rats), Laos (three rats) and Philippines (one rat). In addition, several specimens from four rats in Indonesia were used in the study. Nucleotide sequences of the ribosomal RNA gene (rDNA) (5,649 bp) and the cytochrome c oxidase subunit 1 gene (cox1) (818 bp) were characterised. The rDNA showed little nucleotide variation, including the internal transcribed spacer (ITS) regions. The cox1 showed 24 haplotypes, with up to 15 (1.83%) nucleotide substitutions regardless of parasite origin. Considering that Rattus spp. have been shown to originate from the southern region of Asia and G. neoplasticum is their endogenous parasite, it is reasonable to propose that the present study covers a wide spectrum of the genetic diversity of G. neoplasticum, useful for both the molecular genetic speculation of the species and the molecular genetic differentiation of other local rodent Gongylonema spp. from the cosmopolitan congener.

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis.

PubMed

Blaiotta, Giuseppe; Pepe, Olimpia; Mauriello, Gianluigi; Villani, Francesco; Andolfi, Rosamaria; Moschetti, Giancarlo

2002-12-01

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation.

PubMed

Ehrmann, M A; Vogel, R E

2001-11-01

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.

Gene conversion events and variable degree of homogenization of rDNA loci in cultivars of Brassica napus

PubMed Central

Sochorová, Jana; Coriton, Olivier; Kuderová, Alena; Lunerová, Jana; Chèvre, Anne-Marie; Kovařík, Aleš

2017-01-01

Background and aims Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. Methods We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. Key Results Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH (‘Darmor’, ‘Yudal’ and ‘Asparagus kale’) harboured the same number (12 per diploid set) of loci. In B. napus ‘Darmor’, the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus ‘Darmor’. In contrast, B. napus ‘Yudal’ showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus ‘Asparagus kale’ showed an intermediate pattern to ‘Darmor’ and ‘Yudal’. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar (‘Norin 9’) showed co-dominance. Conclusions The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion. PMID:27707747

DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi.

PubMed

Krüger, Manuela; Stockinger, Herbert; Krüger, Claudia; Schüssler, Arthur

2009-01-01

* At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. * Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. * The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU-ITS-LSU fragment that allows phylogenetic analyses with species level resolution. * The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.

Repetitive DNA loci and their modulation by the non-canonical nucleic acid structures R-loops and G-quadruplexes

PubMed Central

Hall, Amanda C.; Ostrowski, Lauren A.; Mekhail, Karim

2017-01-01

ABSTRACT Cells have evolved intricate mechanisms to maintain genome stability despite allowing mutational changes to drive evolutionary adaptation. Repetitive DNA sequences, which represent the bulk of most genomes, are a major threat to genome stability often driving chromosome rearrangements and disease. The major source of repetitive DNA sequences and thus the most vulnerable constituents of the genome are the rDNA (rDNA) repeats, telomeres, and transposable elements. Maintaining the stability of these loci is critical to overall cellular fitness and lifespan. Therefore, cells have evolved mechanisms to regulate rDNA copy number, telomere length and transposon activity, as well as DNA repair at these loci. In addition, non-canonical structure-forming DNA motifs can also modulate the function of these repetitive DNA loci by impacting their transcription, replication, and stability. Here, we discuss key mechanisms that maintain rDNA repeats, telomeres, and transposons in yeast and human before highlighting emerging roles for non-canonical DNA structures at these repetitive loci. PMID:28406751

A two-locus molecular characterization of Paramecium calkinsi.

PubMed

Przyboś, Ewa; Tarcz, Sebastian; Potekhin, Alexey; Rautian, Maria; Prajer, Małgorzata

2012-03-01

Paramecium calkinsi (Ciliophora, Protozoa) is a euryhaline species which was first identified in freshwater habitats, but subsequently several strains were also collected from brackish water. It is characterized by clockwise spiral swimming movement and the general morphology of the "bursaria type." The present paper is the first molecular characterization of P. calkinsi strains recently collected in distant regions in Russia using ITS1-5.8S- ITS2-5'LSU rDNA (1100bp) and COI (620bp) mtDNA sequenced gene fragments. For comparison, our molecular analysis includes P. bursaria, exhibiting a similar "bursaria morphotype" as well as species representing the "aurelia type," i.e., P. caudatum, P. multimicronucleatum, P. jenningsi, and P. schewiakoffi, and some species of the P. aurelia species complex (P. primaurelia, P. tetraurelia, P. sexaurelia, and P. tredecaurelia). We also use data from GenBank concerning other species in the genus Paramecium and Tetrahymena (which used as an outgroup). The division of the genus Paramecium into four subgenera (proposed by Fokin et al. 2004) is clearly presented by the trees. There is a clear separation between P. calkinsi strains collected from different regions (races). Consequently, given the molecular distances between them, it seems that these races may represent different syngens within the species. Copyright © 2011 Elsevier GmbH. All rights reserved.

Comparative Chloroplast Genome Analyses of Streptophyte Green Algae Uncover Major Structural Alterations in the Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae.

PubMed

Lemieux, Claude; Otis, Christian; Turmel, Monique

2016-01-01

The Streptophyta comprises all land plants and six main lineages of freshwater green algae: Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Charophyceae, Coleochaetophyceae and Zygnematophyceae. Previous comparisons of the chloroplast genome from nine streptophyte algae (including four zygnematophyceans) revealed that, although land plant chloroplast DNAs (cpDNAs) inherited most of their highly conserved structural features from green algal ancestors, considerable cpDNA changes took place during the evolution of the Zygnematophyceae, the sister group of land plants. To gain deeper insights into the evolutionary dynamics of the chloroplast genome in streptophyte algae, we sequenced the cpDNAs of nine additional taxa: two klebsormidiophyceans (Entransia fimbriata and Klebsormidium sp. SAG 51.86), one coleocheatophycean (Coleochaete scutata) and six zygnematophyceans (Cylindrocystis brebissonii, Netrium digitus, Roya obtusa, Spirogyra maxima, Cosmarium botrytis and Closterium baillyanum). Our comparative analyses of these genomes with their streptophyte algal counterparts indicate that the large inverted repeat (IR) encoding the rDNA operon experienced loss or expansion/contraction in all three sampled classes and that genes were extensively shuffled in both the Klebsormidiophyceae and Zygnematophyceae. The klebsormidiophycean genomes boast greatly expanded IRs, with the Entransia 60,590-bp IR being the largest known among green algae. The 206,025-bp Entransia cpDNA, which is one of the largest genome among streptophytes, encodes 118 standard genes, i.e., four additional genes compared to its Klebsormidium flaccidum homolog. We inferred that seven of the 21 group II introns usually found in land plants were already present in the common ancestor of the Klebsormidiophyceae and its sister lineages. At 107,236 bp and with 117 standard genes, the Coleochaete IR-less genome is both the smallest and most compact among the streptophyte algal cpDNAs analyzed thus far; it lacks eight genes relative to its Chaetosphaeridium globosum homolog, four of which represent unique events in the evolutionary scenario of gene losses we reconstructed for streptophyte algae. The 10 compared zygnematophycean cpDNAs display tremendous variations at all levels, except gene content. During zygnematophycean evolution, the IR disappeared a minimum of five times, the rDNA operon was broken at four distinct sites, group II introns were lost on at least 43 occasions, and putative foreign genes, mainly of phage/viral origin, were gained.

Comparative Chloroplast Genome Analyses of Streptophyte Green Algae Uncover Major Structural Alterations in the Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae

PubMed Central

Lemieux, Claude; Otis, Christian; Turmel, Monique

2016-01-01

The Streptophyta comprises all land plants and six main lineages of freshwater green algae: Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Charophyceae, Coleochaetophyceae and Zygnematophyceae. Previous comparisons of the chloroplast genome from nine streptophyte algae (including four zygnematophyceans) revealed that, although land plant chloroplast DNAs (cpDNAs) inherited most of their highly conserved structural features from green algal ancestors, considerable cpDNA changes took place during the evolution of the Zygnematophyceae, the sister group of land plants. To gain deeper insights into the evolutionary dynamics of the chloroplast genome in streptophyte algae, we sequenced the cpDNAs of nine additional taxa: two klebsormidiophyceans (Entransia fimbriata and Klebsormidium sp. SAG 51.86), one coleocheatophycean (Coleochaete scutata) and six zygnematophyceans (Cylindrocystis brebissonii, Netrium digitus, Roya obtusa, Spirogyra maxima, Cosmarium botrytis and Closterium baillyanum). Our comparative analyses of these genomes with their streptophyte algal counterparts indicate that the large inverted repeat (IR) encoding the rDNA operon experienced loss or expansion/contraction in all three sampled classes and that genes were extensively shuffled in both the Klebsormidiophyceae and Zygnematophyceae. The klebsormidiophycean genomes boast greatly expanded IRs, with the Entransia 60,590-bp IR being the largest known among green algae. The 206,025-bp Entransia cpDNA, which is one of the largest genome among streptophytes, encodes 118 standard genes, i.e., four additional genes compared to its Klebsormidium flaccidum homolog. We inferred that seven of the 21 group II introns usually found in land plants were already present in the common ancestor of the Klebsormidiophyceae and its sister lineages. At 107,236 bp and with 117 standard genes, the Coleochaete IR-less genome is both the smallest and most compact among the streptophyte algal cpDNAs analyzed thus far; it lacks eight genes relative to its Chaetosphaeridium globosum homolog, four of which represent unique events in the evolutionary scenario of gene losses we reconstructed for streptophyte algae. The 10 compared zygnematophycean cpDNAs display tremendous variations at all levels, except gene content. During zygnematophycean evolution, the IR disappeared a minimum of five times, the rDNA operon was broken at four distinct sites, group II introns were lost on at least 43 occasions, and putative foreign genes, mainly of phage/viral origin, were gained. PMID:27252715

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

NASA Technical Reports Server (NTRS)

Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

1990-01-01

The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

PubMed Central

Perkins, David D.; Metzenberg, Robert L.; Raju, Namboori B.; Selker, Eric U.; Barry, Edward G.

1986-01-01

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered. PMID:2947829

Oligonucleotide fingerprinting of rRNA genes for analysis of fungal community composition.

PubMed

Valinsky, Lea; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

2002-12-01

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated with Ascomycota, while 12% belonged to BASIDIOMYCOTA: A large fraction of the clones were affiliated with the genera Fusarium (404 clones) and Raciborskiomyces (176 clones). Smaller assemblages of clones had high sequence identities to the Alternaria, Ascobolus, Chaetomium, Cryptococcus, and Rhizoctonia clades.

Identification of Giardia species and Giardia duodenalis assemblages by sequence analysis of the 5.8S rDNA gene and internal transcribed spacers.

PubMed

Cacciò, Simone M; Beck, Relja; Almeida, Andre; Bajer, Anna; Pozio, Edoardo

2010-05-01

PCR assays have been developed mainly to assist investigations into the epidemiology of Giardia duodenalis, the only species in the Giardia genus having zoonotic potential. However, a reliable identification of all species is of practical importance, particularly when water samples and samples from wild animals are investigated. The aim of the present work was to genotype Giardia species and G. duodenalis assemblages using as a target the region spanning the 5.8S gene and the 2 flanking internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene. Primers were designed to match strongly conserved regions in the 3' end of the small subunit and in the 5' end of the large subunit ribosomal genes. The corresponding region (about 310 bp) was amplified from 49 isolates of both human and animal origin, representing all G. duodenalis assemblages as well as G. muris and G. microti. Sequence comparison and phylogenetic analysis showed that G. ardeae, G. muris, G. microti as well as the 7 G. duodenalis assemblages can be easily distinguished. Since the major subgroups within the zoonotic assemblages A and B can be identified by sequence analysis, this assay is also informative for molecular epidemiological studies.

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line

PubMed Central

Mondin, Mateus; Santos-Serejo, Janay A.; Bertäo, Mônica R.; Laborda, Prianda; Pizzaia, Daniel; Aguiar-Perecin, Margarida L. R.

2014-01-01

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA. PMID:25352856

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

PubMed Central

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-01-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

Bacillus pumilus SAFR-032 isolate

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri J. (Inventor)

2007-01-01

The present invention relates to discovery and isolation of a biologically pure culture of a Bacillus pumilus SAFR-032 isolate with UV sterilization resistant properties. This novel strain has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus. The GenBank accession number for the 16S rDNA sequence of the Bacillus pumilus SAFR-032 isolate is AY167879.

Uncovering the molecular organization of unusual highly scattered 5S rDNA: The case of Chariesterus armatus (Heteroptera).

PubMed

Bardella, Vanessa Bellini; Cabral-de-Mello, Diogo Cavalcanti

2018-03-10

One cluster of 5S rDNA per haploid genome is the most common pattern among Heteroptera. However, in Chariesterus armatus, highly scattered signals were noticed. We isolated and characterized the entire 5S rDNA unit of C. armatus aiming to a deeper knowledge of molecular organization of the 5S rDNA among Heteroptera and to understand possible causes and consequences of 5S rDNA chromosomal spreading. For a comparative analysis, we performed the same approach in Holymenia histrio with 5S rDNA restricted to one bivalent. Multiple 5S rDNA variants were observed in both species, though they were more variable in C. armatus, with some of variants corresponding to pseudogenes. These pseudogenes suggest birth-and-death mechanism, though homogenization was also observed (concerted evolution), indicating evolution through mixed model. Association between transposable elements and 5S rDNA was not observed, suggesting spreading of 5S rDNA through other mechanisms, like ectopic recombination. Scattered organization is a rare example for 5S rDNA, and such organization in C. armatus genome could have led to the high diversification of sequences favoring their pseudogenization. Copyright © 2017. Published by Elsevier B.V.

Reference karyotype and cytomolecular map for loblolly pine (Pinus taeda L.)

Treesearch

M. Nurul Islam-faridi; C. Dana Nelson; Thomas L. Kubisiak

2007-01-01

A reference karyotype is presented for loblolly pine (Pinus taeda L., subgenus Pinus , section Pinus, subsection Australes), based on fluorescent in situ hybridization (FISH), using 18s-28s rDNA, 5s rDNA, and Arabidopsis-type telomere repeat sequence (A-type TRS). Well...

Genetic characterization and phylogenetic analysis of Eimeria arloingi in Iranian native kids.

PubMed

Khodakaram-Tafti, A; Hashemnia, M; Razavi, S M; Sharifiyazdi, H; Nazifi, S

2013-09-01

Among the 16 species of Eimeria from goats, Eimeria arloingi and Eimeria ninakohlyakimovae are regarded as the most pathogenic species in the world and cause clinical caprine coccidiosis. E. arloingi is known to be an important cause of coccidiosis in Iranian kids. Molecular analyses of two portions of nuclear ribosomal DNA (internal transcribed spacer1 (ITS1) and 18S rDNA) were used for the genetic characterization of the E. arloingi. Comparison of the sequencing data of E. arloingi obtained in the present study (ITS1: KC507793 and 18S rDNA: KC507792) with other Eimeria species in the GenBank database revealed a particularly close relationship between E. arloingi and Eimeria spp. from the cattle and sheep. The phylogram based on the ITS1 sequences shows that the E. arloingi, Eimeria bovis, and Eimeria zuernii formed a distinct group separate from the other remaining Eimeria spp. in cattle and poultry. In pairwise alignment, 18S rDNA sequence derived from E. arloingi showed 99% similarity to Eimeria ahsata with differences observed at only three nucleotides. This study showed that the ITS1 and 18S rDNA gene are useful genetic markers for the specific identification and differentiation of Eimeria spp. in ruminants.

PCR tools for the verification of the specific identity of ascaridoid nematodes from dogs and cats.

PubMed

Li, M W; Lin, R Q; Chen, H H; Sani, R A; Song, H Q; Zhu, X Q

2007-01-01

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of Toxocara canis, Toxocara cati, Toxocara malaysiensis and Toxascaris leonina, specific forward primers were designed in the ITS-1 or ITS-2 for each of the four ascaridoid species of dogs and cats. These primers were used individually together with a conserved primer in the large subunit of rDNA to amplify partial ITS-1 and/or ITS-2 of rDNA from 107 DNA samples from ascaridoids from dogs and cats in China, Australia, Malaysia, England and the Netherlands. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amounts of DNA detectable using the PCR assays were 0.13-0.54ng. These PCR assays should provide useful tools for the diagnosis and molecular epidemiological investigations of toxocariasis in humans and animals.

Species-specific identification of commercial probiotic strains.

PubMed

Yeung, P S M; Sanders, M E; Kitts, C L; Cano, R; Tong, P S

2002-05-01

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales.

PubMed

Richert, Kathrin; Brambilla, Evelyne; Stackebrandt, Erko

2005-01-01

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.

Curiously modern DNA for a "250 million-year-old" bacterium.

PubMed

Nickle, David C; Learn, Gerald H; Rain, Matthew W; Mullins, James I; Mittler, John E

2002-01-01

Studies of ancient DNA have attracted considerable attention in scientific journals and the popular press. Several of the more extreme claims for ancient DNA have been questioned on biochemical grounds (i.e., DNA surviving longer than expected) and evolutionary grounds (i.e., nucleotide substitution patterns not matching theoretical expectations for ancient DNA). A recent letter to Nature from Vreeland et al. (2000), however, tops all others with respect to age and condition of the specimen. These researchers extracted and cultured a bacterium from an inclusion body from what they claim is a 250 million-year (Myr)-old salt crystal. If substantiated, this observation could fundamentally alter views about bacterial physiology, ecology and evolution. Here we report on molecular evolutionary analyses of the 16S rDNA from this specimen. We find that 2-9-3 differs from a modern halophile, Salibacillus marismortui, by just 3 unambiguous bp in 16S rDNA, versus the approximately 59 bp that would be expected if these bacteria evolved at the same rate as other bacteria. We show, using a Poisson distribution, that unless it can be shown that S. marismortui evolves 5 to 10 times more slowly than other bacteria for which 16S rDNA substitution rates have been established, Vreeland et al.'s claim would be rejected at the 0.05 level. Also, a molecular clock test and a relative rates test fail to substantiate Vreeland et al.'s claim that strain 2-9-3 is a 250-Myr-old bacterium. The report of Vreeland et al. thus falls into a long series of suspect ancient DNA studies.

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries.

PubMed

Ko, Kwan Soo; Oh, Won Sup; Peck, Kyong Ran; Lee, Jang Ho; Lee, Nam Yong; Song, Jae-Hoon

2005-07-01

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.

[Molecular genetic characterization of the Far Eastern trematode Skrjabino lecithum spasskii Belous, 1954 (Digenea: Haploporidae)), a parasite of mullets].

PubMed

Atopkin, D M; Nikitenko, A Yu; Ngo, H D; Ha, N V; Tang, N V

2015-01-01

Intraspecific genetic differentiation of the trematode Skrjabinolecithum spasskii and its phylogenetic relationships with other species of the family Haploporidae were studied by comparing the nucleotide sequences of a part of the 28S rRNA gene and the ITS1-5.8S-ITS2 rDNA region. Trematodes were isolated from so-iuy mullet Liza haematocheila fishes collected in rivers of Primorye and flathead grey mullet Mugil cephalus fishes collected in water bodies of Vietnam (27 fishes in total). A phylogenetic analysis showed that S. spasskii is close to species of the genus Capitimitta of the subfamily Waretrematinae. By intraspecific variation of rDNA sequences, trematodes were divided into three groups with tree different genotypes, which had fixed nucleotide substitutions. Genotype I was found in trematodes from fishes collected in Primorye. Genotype II was detected in trematodes from M. cephalus fishes collected in the Tonkin Bay, Cat Ba Island, Vietnam. Genotype III was found in five trematodes from L. haematocheila collected in the Kievka River, Primorye. The genetic distances between genotypes I and III from Primorye were 0.4 and 0.65% by 28S and ITS rDNA sequences, respectively. The lowest genetic distances were observed between genotypes II (Vietnam) and III (Primorye), 0.1 and 0.33% by 28S and ITS rDNA sequences, respectively. Possible causes of genetic differentiation of S. spasskii from different geographic locations and different definitive host species are discussed.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

PubMed

Decelle, Johan; Romac, Sarah; Stern, Rowena F; Bendif, El Mahdi; Zingone, Adriana; Audic, Stéphane; Guiry, Michael D; Guillou, Laure; Tessier, Désiré; Le Gall, Florence; Gourvil, Priscillia; Dos Santos, Adriana L; Probert, Ian; Vaulot, Daniel; de Vargas, Colomban; Christen, Richard

2015-11-01

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing. © 2015 John Wiley & Sons Ltd.

Morphological and molecular characterisation of Myxobolus pronini n. sp. (Myxozoa: Myxobolidae) from the abdominal cavity and visceral serous membranes of the gibel carp Carassius auratus gibelio (Bloch) in Russia and China.

PubMed

Liu, Xin-Hua; Batueva, Marina-D; Zhao, Yuan-Li; Zhang, Jin-Yong; Zhang, Qian-Qian; Li, Tong-Tong; Li, Ai-Hua

2016-10-25

Myxozoa is a well-known economically and ecologically important group of metazoan parasites, phylogenetically related to Cnidaria. High diversity of myxosporeans has been recorded in Russia and China; however, most of the species were solely morphologically characterised. Here, we identified a new gibel carp-infecting Myxobolus species and morphologically and molecularly compared the Russian and Chinese isolates of this new myxosporean. Myxobolus pronini n. sp. was found free in the abdominal cavity of Carassius auratus gibelio (Bloch, 1782) in Lake Baikal watershed, Russia, and embedded in the visceral serous membranes of the same fish species in Lake Taibai, Hubei province, China. The morphometric data of the plasmodia and mature spores exhibited some differences between the Russian and Chinese isolates, but SSU rDNA sequences indicated that these two geographical isolates are conspecific. The mature spores from the two locations are obovate in frontal view, with wider anterior than posterior end and lemon-shaped in sutural view. Spores of the Russian isolate were 14.3-16.2 (mean 15.1 ± 0.2) μm long, 9.6-10.8 (10.1 ± 0.1) μm wide and 6.4-7.4 (6.7 ± 0.15) μm thick; those of the Chinese isolate were 13.8-15.6 (14.7 ± 0.24) μm long, 9.6-13.3 (9.6 ± 0.65) μm wide and 6.2-7.2 (6.6 ± 0.16) μm thick. The newly-generated rDNA sequences (including SSU rDNA, ITS and LSU rDNA) from the two isolates represented some variations within the intraspecific range. Homology search by BLAST showed that the newly obtained rDNA sequences do not match any sequences available on GenBank. Phylogenetic analysis based on the aligned partial SSU rDNA sequences indicated that this novel species clustered with several gibel carp-infecting Myxobolus spp. with round anterior end of spores. Additionally, phylogenetic analysis based on all obtained ITS sequences showed that distinct genetic geographical differentiation occurred for this new parasite. Myxobolus pronini n. sp. is described by integrating morphological, ecological and molecular evidence. Two geographical isolates of this species showed some morphological and genetic differences but within the intraspecific range of variation.

Molecular phylogeny of the lionfish genera Dendrochirus and Pterois (Scorpaenidae, Pteroinae) based on mitochondrial DNA sequences.

PubMed

Kochzius, Marc; Söller, Rainer; Khalaf, Maroof A; Blohm, Dietmar

2003-09-01

This study investigates the molecular phylogeny of seven lionfishes of the genera Dendrochirus and Pterois. MP, ML, and NJ phylogenetic analysis based on 964 bp of partial mitochondrial DNA sequences (cytochrome b and 16S rDNA) revealed two main clades: (1) "Pterois" clade (Pterois miles and Pterois volitans), and (2) "Pteropterus-Dendrochirus" clade (remainder of the sampled species). The position of Dendrochirus brachypterus either basal to the main clades or in the "Pteropterus-Dendrochirus" clade cannot be resolved. However, the molecular phylogeny did not support the current separation of the genera Pterois and Dendrochirus. The siblings P. miles and P. volitans are clearly separated and our results support the proposed allopatric or parapatric distribution in the Indian and Pacific Ocean. However, the present analysis cannot reveal if P. miles and P. volitans are separate species or two populations of a single species, because the observed separation in different clades can be either explained by speciation or lineage sorting. Molecular clock estimates for the siblings P. miles and P. volitans suggest a divergence time of 2.4-8.3 mya, which coincide with geological events that created vicariance between populations of the Indian and Pacific Ocean.

Archaeal Diversity in Waters from Deep South African Gold Mines

PubMed Central

Takai, Ken; Moser, Duane P.; DeFlaun, Mary; Onstott, Tullis C.; Fredrickson, James K.

2001-01-01

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated. PMID:11722932

Ribosomal DNA, tri- and bi-partite pericentromeres in the permanent translocation heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Szklarczyk, Marek

2010-12-01

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A(3)-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.

Molecular Characterization and Analysis of 16S Ribosomal DNA in Some Isolates of Demodex folicullorum

PubMed Central

DANESHPARVAR, Afrooz; MOWLAVI, Gholamreza; MIRJALALI, Hamed; HAJJARAN, Homa; MOBEDI, Iraj; NADDAF, Saeed Reza; SHIDFAR, Mohammadreza; SADAT MAKKI, Mahsa

2017-01-01

Background: Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct and transmits through close contact with an infested host. Methods: This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, already obtained from four different patients and identified morphologically though clearing with 10% Potassium hydroxide (KOH) and microscopical examination. Amplified fragments from the isolates were compared with GeneBank database and phylogenetic analysis was carried out using MEGA6 software. Results: A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs) in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Conclusion: Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites. PMID:28761482

Molecular Characterization and Analysis of 16S Ribosomal DNA in Some Isolates of Demodex folicullorum.

PubMed

Daneshparvar, Afrooz; Mowlavi, Gholamreza; Mirjalali, Hamed; Hajjaran, Homa; Mobedi, Iraj; Naddaf, Saeed Reza; Shidfar, Mohammadreza; Sadat Makki, Mahsa

2017-01-01

Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct and transmits through close contact with an infested host. This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, already obtained from four different patients and identified morphologically though clearing with 10% Potassium hydroxide (KOH) and microscopical examination. Amplified fragments from the isolates were compared with GeneBank database and phylogenetic analysis was carried out using MEGA6 software. A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs) in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites.

Detection and Phylogenetic Analysis of Wolbachia in the Asiatic Rice Leafroller, Cnaphalocrocis medinalis, in Chinese Populations

PubMed Central

Chai, Huan-Na; Du, Yu-Zhou; Qiu, Bao-Li; Zhai, Bao-Ping

2011-01-01

Wolbachia are a group of intracellular inherited endosymbiontic bacteria infecting a wide range of insects. In this study the infection status of Wolbachia (Rickettsiales: Rickettsiaceae) was measured in the Asiatic rice leafroller, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), from twenty locations in China by sequencing wsp, ftsZ and 16S rDNA genes. The results showed high infection rates of Wolbachia in C. medinalis populations. Wolbachia was detected in all geographically separate populations; the average infection rate was ∼ 62.5%, and the highest rates were 90% in Wenzhou and Yangzhou populations. The Wolbachia detected in different C. medinalis populations were 100% identical to each other when wsp, ftsZ, and 16S rDNA sequences were compared, with all sequences belonging to the Wolbachia B supergroup. Based on wsp, ftsZ and 16S rDNA sequences of Wolbachia, three phylogenetic trees of similar pattern emerged. This analysis indicated the possibility of inter-species and intra-species horizontal transmission of Wolbachia in different arthropods in related geographical regions. The migration route of C. medinalis in mainland China was also discussed since large differentiation had been found between the wsp sequences of Chinese and Thai populations. PMID:22233324

Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

PubMed Central

Takahashi, Shunsuke; Tomita, Junko; Nishioka, Kaori; Hisada, Takayoshi; Nishijima, Miyuki

2014-01-01

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples. PMID:25144201

[Detection and diversity analysis of rumen methanogens in the co-cultures with anaerobic fungi].

PubMed

Cheng, Yan-fen; Mao, Sheng-yong; Pei, Cai-xia; Liu, Jian-xin; Zhu, Wei-yun

2006-12-01

Rumen methanogen diversity in the co-cultures with anaerobic fungi from goat rumen was analyzed. Mix-cultures of anaerobic fungi and methanogens were obtained from goat rumen using anaerobic fungal medium and the addition of penicillin and streptomycin and then subcultured 62 times by transferring cultures every 3 - 4d. Total DNA from the original rumen fluid and subcultured fungal cultures was used for PCR/DGGE and RFLP analysis. 16S rDNA of clones corresponding to representative OTUs were sequenced. Results showed that the diversity index (Shannon index) of the methanogens generated from DGGE profiles reduced from 1.32 to 0.99 from rumen fluid to fungal culture after 45 subculturing, with the lowest similarity of DGGE profiles at 34.7%. The Shannon index increased from 0.99 to 1.15 from the fungal culture after 45 subculturing to that after 62 subculturing, with the lowest similarity at 89.2% . A total of 5 OTUs were obtained from 69. clones using RFLP analysis and six clones representing the 5 OTUs respectively were sequenced. Of the 5 OTUs, three had their cloned 16S rDNA sequences most closely related to uncultured archaeal symbiont PA202 with the same similarity of 95 %, but had not closely related to any identified culturable methanogen. The rest two OTUs had their cloned 16S rDNA sequences sharing the same closest relative, uncultured rumen methanogen 956, with the same similarity of 97% .Their 16S rDNA sequences of these two OTUs also showed 97% similar to the closest identified culturable methanogen Methanobrevibacter sp. NT7. In conclusion, diverse yet unidentified rumen methanogen species exist in the co-cultures with anaerobic fungi isolated from the goat rumen.

Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae.

PubMed

Lim, K Yoong; Kovarik, Ales; Matyasek, Roman; Chase, Mark W; Knapp, Sandra; McCarthy, Elizabeth; Clarkson, James J; Leitch, Andrew R

2006-12-01

Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.

Evidence for 5S rDNA horizontal transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families.

PubMed

Merlo, Manuel A; Cross, Ismael; Palazón, José L; Ubeda-Manzanaro, María; Sarasquete, Carmen; Rebordinos, Laureana

2012-10-07

The Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH). Two types of 5S rDNA were observed, named type α and type β. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type β specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species. A highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology.

Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact

PubMed Central

Zagoskin, Maxim V.; Lazareva, Valentina I.; Grishanin, Andrey K.; Mukha, Dmitry V.

2014-01-01

The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals. PMID:25215300

Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China

NASA Astrophysics Data System (ADS)

Zhou, Hong; Zhang, Zhinan; Chen, Haiyan; Sun, Renhua; Wang, Hui; Guo, Lei; Pan, Haijian

2010-07-01

In this study, we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes. Using 16S rDNA as a complementary marker and combining morphological and ecological characterization, some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status. We obtained 22 haplotype gene sequences of 13 taxa, including 10 CO1 sequences and 12 16S rDNA sequences. Based on intra- and inter-specific distances, we built phylogenetic trees using the neighbor-joining method. Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes, but other genes, such as 16S rDNA, could be used as a complementary genetic marker. For more accurate species identification and effective testing of species hypothesis, DNA barcoding should be incorporated with morphological, ecological, biogeographical, and phylogenetic information. The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.

Comparison of the ITS1 and ITS2 rDNA in Emeria callospermophili (Apicomplexa: Eimeriidae) from Sciurid Rodents

PubMed Central

Motriuk-Smith, Dagmara; Seville, R Scott; Quealy, Leah; Oliver, Clinton E.

2011-01-01

The taxonomy of the coccidia has historically been morphologically based. The purpose of this study was to establish if conspecificity of isolates of Eimeria callospermophili from 4 ground-dwelling squirrel hosts (Rodentia: Sciuridae) is supported by comparison of rDNA sequence data and to examine how this species relates to eimerian species from other sciurid hosts. Eimeria callospermophili was isolated from 4 wild caught hosts, i.e., Urocitellus elegans, Cynomys leucurus, Marmota flaviventris, and Cynomys ludovicianus. The ITS1 and ITS2 genomic rDNA sequences were PCR generated, sequenced, and analyzed. The highest intraspecific pairwise distance values of 6.0% in ITS1 and 7.1% in ITS2 were observed in C. leucurus. Interspecific pairwise distance values greater than 5% do not support E. callospermophili conspecificity. Generated E. callospermophili sequences were compared to Eimeria lancasterensis from Sciuris niger and Sciurus niger cinereus, and Eimeria ontarioensis from S. niger. A single well-supported clade was formed by E. callospermophili amplicons in Neighbor Joining and Maximum Parsimony analyses. However, within the clade there was little evidence of host or geographic structuring of the species. PMID:21506777

Phylogenetic relationships in Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA partial sequences.

PubMed

Zhao, Ya-E; Wu, Li-Ping

2012-09-01

To confirm phylogenetic relationships in Demodex mites based on mitochondrial 16S rDNA partial sequences, mtDNA 16S partial sequences of ten isolates of three Demodex species from China were amplified, recombined, and sequenced and then analyzed with two Demodex folliculorum isolates from Spain. Lastly, genetic distance was computed, and phylogenetic tree was reconstructed. MEGA 4.0 analysis showed high sequence identity among 16S rDNA partial sequences of three Demodex species, which were 95.85 % in D. folliculorum, 98.53 % in Demodex canis, and 99.71 % in Demodex brevis. The divergence, genetic distance, and transition/transversions of the three Demodex species reached interspecies level, whereas there was no significant difference of the divergence (1.1 %), genetic distance (0.011), and transition/transversions (3/1) of the two geographic D. folliculorum isolates (Spain and China). Phylogenetic trees reveal that the three Demodex species formed three separate branches of one clade, where D. folliculorum and D. canis gathered first, and then gathered with D. brevis. The two Spain and five China D. folliculorum isolates did not form sister clades. In conclusion, 16S mtDNA are suitable for phylogenetic relationship analysis in low taxa (genus or species), but not for intraspecies determination of Demodex. The differentiation among the three Demodex species has reached interspecies level.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of pakistan and their industrial importance

PubMed Central

Akhtar, Nasrin; Ghauri, Muhammad A.; Iqbal, Aamira; Anwar, Munir A.; Akhtar, Kalsoom

2008-01-01

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications. PMID:24031194

Introducing W.A.T.E.R.S.: a workflow for the alignment, taxonomy, and ecology of ribosomal sequences.

PubMed

Hartman, Amber L; Riddle, Sean; McPhillips, Timothy; Ludäscher, Bertram; Eisen, Jonathan A

2010-06-12

For more than two decades microbiologists have used a highly conserved microbial gene as a phylogenetic marker for bacteria and archaea. The small-subunit ribosomal RNA gene, also known as 16 S rRNA, is encoded by ribosomal DNA, 16 S rDNA, and has provided a powerful comparative tool to microbial ecologists. Over time, the microbial ecology field has matured from small-scale studies in a select number of environments to massive collections of sequence data that are paired with dozens of corresponding collection variables. As the complexity of data and tool sets have grown, the need for flexible automation and maintenance of the core processes of 16 S rDNA sequence analysis has increased correspondingly. We present WATERS, an integrated approach for 16 S rDNA analysis that bundles a suite of publicly available 16 S rDNA analysis software tools into a single software package. The "toolkit" includes sequence alignment, chimera removal, OTU determination, taxonomy assignment, phylogentic tree construction as well as a host of ecological analysis and visualization tools. WATERS employs a flexible, collection-oriented 'workflow' approach using the open-source Kepler system as a platform. By packaging available software tools into a single automated workflow, WATERS simplifies 16 S rDNA analyses, especially for those without specialized bioinformatics, programming expertise. In addition, WATERS, like some of the newer comprehensive rRNA analysis tools, allows researchers to minimize the time dedicated to carrying out tedious informatics steps and to focus their attention instead on the biological interpretation of the results. One advantage of WATERS over other comprehensive tools is that the use of the Kepler workflow system facilitates result interpretation and reproducibility via a data provenance sub-system. Furthermore, new "actors" can be added to the workflow as desired and we see WATERS as an initial seed for a sizeable and growing repository of interoperable, easy-to-combine tools for asking increasingly complex microbial ecology questions.

Physical locations of 5S and 18S-25S rDNA in Asian and American diploid Hordeum species with the I genome.

PubMed

Taketa, S; Ando, H; Takeda, K; von Bothmer, R

2001-05-01

The physical locations of 5S and 18S-25S rDNA sequences in 15 diploid Hordeum species with the I genome were examined by double-target in situ hybridization with pTa71 (18S-25S rDNA) and pTa794 (5S rDNA) clones as probes. All the three Asian species had a species-specific rDNA pattern. In 12 American species studied, eight different rDNA types were found. The type reported previously in H. chilense (the 'chilense' type) was observed in eight American species. The chilense type had double 5S rDNA sites - two sites on one chromosome arm separated by a short distance - and two pairs of major 18S-25S rDNA sites on two pairs of satellite chromosomes. The other seven types found in American species were similar to the chilense type and could be derived from the chilense type through deletion, reduction or addition of a rDNA site. Intraspecific polymorphisms were observed in three American species. The overall similarity in rDNA patterns among American species indicates the close relationships between North and South American species and their derivation from a single ancestral source. The differences in the distribution patterns of 5S and 18S-25S rDNA between Asian and American species suggest differentiation between the I genomes of Asian and American species. The 5S and 18S-25S rDNA sites are useful chromosome markers for delimiting Asian species, but have limited value as a taxonomic character in American species. On the basis of rDNA patterns, karyotype evolution and phylogeny of the I-genome diploid species are discussed.

Strain diversity and host specificity in bee gut symbionts revealed by deep sampling of single copy protein-coding sequences

PubMed Central

Powell, J. Elijah; Ratnayeke, Nalin; Moran, Nancy A.

2017-01-01

High throughput rRNA amplicon surveys of bacterial communities provide a rapid snapshot of taxonomic composition. But strains with nearly identical rRNA sequences often differ in gene repertoires and metabolic capabilities. To assess strain-level variation within Snodgrassella alvi, a gut symbiont of corbiculate bees, we performed deep sequencing on amplicons of a single copy coding gene (minD) as well as the 16S rDNA V4 region. We surveyed honey bees (Apis mellifera) sampled globally and 12 bumble bee species (Bombus) sampled from two regions of the USA. The minD analyses reveal that S. alvi contains far more strain diversity than is evident from 16S rDNA analysis. Many taxa inferred on the basis of 16S rDNA are shared between A. mellifera and Bombus species, but taxa inferred on the basis of minD are never shared and often are restricted to particular Bombus species. Clustering based on minD revealed that gut communities often reflect host species and geographic location. Both minD and 16S rDNA analyses indicate that strain diversity is higher in A. mellifera than in Bombus species. The minD locus flanks a 16S gene, enabling development of strain-specific 16S fluorescent probes to illuminate the spatial relationship of strains within the bee gut. PMID:27482856

Discrimination of closely related species in tintinnid ciliates: new insights on crypticity and polymorphism in the genus Helicostomella.

PubMed

Santoferrara, Luciana F; Tian, Michael; Alder, Viviana A; McManus, George B

2015-02-01

This study focuses on the utility of molecular markers for the discrimination of closely related species in tintinnid ciliates. We analyzed the ecologically important genus Helicostomella by sequencing part of the large-subunit rDNA (LSU rDNA) and the 5.8S rDNA combined with the internally transcribed spacer regions 1 and 2 (5.8S rDNA-ITS) from forty-five individuals collected in NW and SW Atlantic waters and after culturing. Although all described Helicostomella species represent a continuum of morphologies, forms with shorter or longer loricae would correspond to different species according to previous molecular data. Here we observed that long forms show both crypticity (i.e. two almost identical long forms with different DNA sequences) and polymorphism (i.e. some long forms develop significantly shorter loricae after culturing). Reviewing all available tintinnid sequences, we found that 1) three Helicostomella clusters are consistent with different species from a molecular perspective, although these clusters are neither clearly differentiated by their loricae nor unambiguously linked to described species, 2) Helicostomella is closely related (probably to the family or genus level) to four "Tintinnopsis-like" morphospecies, and 3) if considered separately, neither LSU rDNA nor 5.8S rDNA-ITS completely discriminate closely related species, thus supporting the use of multi-gene barcodes for tintinnids. Copyright © 2014 Elsevier GmbH. All rights reserved.

Characteristic component profiling and identification of different Uncaria species based on high-performance liquid chromatography-photodiode array detection tandem ion trap and time of flight mass spectrometry coupled with rDNA ITS sequence.

PubMed

Zhao, Bingqiang; Huang, Yanjun; Chen, Qiulan; Chen, Qizhao; Miao, Hui; Zhu, Shuang; Zeng, Changqing

2018-03-01

Uncaria is a multi-source herb and its species identification has become a bottleneck in quality control. To study the identification method of different Uncaria species herbs through HPLC-MS coupled with rDNA Internal Transcribed Spacer (rDNA ITS) sequence, both plant morphological traits and molecular identification were used to determine the species of every collected Uncaria herb. The genetic analysis of different Uncaria species was performed using their rDNA ITS sequence as a molecular marker. Meanwhile, the phylogenetic relationships of 22 samples from six Uncaria species were divided and classified clearly. By optimizing the chromatographic conditions, a practical HPLC method to differentiate various varieties of Uncaria herbs was set up based on a set of characteristic components across each species. A high-performance liquid chromatography-photodiode array detector tandem ion trap and time of flight mass spectrometry technique combined with reference substances was utilized to derive 21 characteristic compounds containing six groups of six Uncaria species in China. Thus, this study provides a feasible method to solve the current problem of confusion in Uncaria species, and makes a significant step forward in the appropriate clinical use, in-depth research and further utilization of different Uncaria species. Copyright © 2017 John Wiley & Sons, Ltd.

Detection of Hepatozoon felis in Ticks Collected from Free-Ranging Amur Tigers ( Panthera tigris altaica), Russian Far East, 2002-12.

PubMed

Thomas, Lindsay H; Seryodkin, Ivan V; Goodrich, John M; Miquelle, Dale G; Birtles, Richard J; Lewis, John C M

2016-07-01

We collected 69 ticks from nine, free-ranging Amur tigers ( Panthera tigris altaica) between 2002 and 2011 and investigated them for tick-borne pathogens. DNA was extracted using alkaline digestion and PCR was performed to detect apicomplexan organisms. Partial 18S rDNA amplification products were obtained from 14 ticks from four tigers, of which 13 yielded unambiguous nucleotide sequence data. Comparative sequence analysis revealed all 13 partial 18S rDNA sequences were most similar to those belonging to strains of Hepatozoon felis (>564/572 base-pair identity, >99% sequence similarity). Although this tick-borne protozoon pathogen has been detected in wild felids from many parts of the world, this is the first record from the Russian Far East.

Phylogeny and evolution of ferns (monilophytes) with a focus on the early leptosporangiate divergences.

PubMed

Pryer, Kathleen M; Schuettpelz, Eric; Wolf, Paul G; Schneider, Harald; Smith, Alan R; Cranfill, Raymond

2004-10-01

The phylogenetic structure of ferns (= monilophytes) is explored here, with a special focus on the early divergences among leptosporangiate lineages. Despite considerable progress in our understanding of fern relationships, a rigorous and comprehensive analysis of the early leptosporangiate divergences was lacking. Therefore, a data set was designed here to include critical taxa that were not included in earlier studies. More than 5000 bp from the plastid (rbcL, atpB, rps4) and the nuclear (18S rDNA) genomes were sequenced for 62 taxa. Phylogenetic analyses of these data (1) confirm that Osmundaceae are sister to the rest of the leptosporangiates, (2) resolve a diverse set of ferns formerly thought to be a subsequent grade as possibly monophyletic (((Dipteridaceae, Matoniaceae), Gleicheniaceae), Hymenophyllaceae), and (3) place schizaeoid ferns as sister to a large clade of "core leptosporangiates" that includes heterosporous ferns, tree ferns, and polypods. Divergence time estimates for ferns are reported from penalized likelihood analyses of our molecular data, with constraints from a reassessment of the fossil record.

DNA microarrays for identifying fishes.

PubMed

Kochzius, M; Nölte, M; Weber, H; Silkenbeumer, N; Hjörleifsdottir, S; Hreggvidsson, G O; Marteinsson, V; Kappel, K; Planes, S; Tinti, F; Magoulas, A; Garcia Vazquez, E; Turan, C; Hervet, C; Campo Falgueras, D; Antoniou, A; Landi, M; Blohm, D

2008-01-01

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a "Fish Chip" for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

16S rDNA-based metagenomic analysis of dental plaque and lung bacteria in patients with severe acute exacerbations of chronic obstructive pulmonary disease.

PubMed

Tan, L; Wang, H; Li, C; Pan, Y

2014-12-01

Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are leading causes of mortality in hospital intensive care units. We sought to determine whether dental plaque biofilms might harbor pathogenic bacteria that can eventually cause lung infections in patients with severe AE-COPD. Paired samples of subgingival plaque biofilm and tracheal aspirate were collected from 53 patients with severe AE-COPD. Total bacterial DNA was extracted from each sample individually for polymerase chain reaction amplification and/or generation of bacterial 16S rDNA sequences and cDNA libraries. We used a metagenomic approach, based on bacterial 16S rDNA sequences, to compare the distribution of species present in dental plaque and lung. Analysis of 1060 sequences (20 clones per patient) revealed a wide range of aerobic, anaerobic, pathogenic, opportunistic, novel and uncultivable bacterial species. Species indistinguishable between the paired subgingival plaque and tracheal aspirate samples (97-100% similarity in 16S rDNA sequence) were dental plaque pathogens (Aggregatibacter actinomycetemcomitans, Capnocytophaga sputigena, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and lung pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumoniae). Real-time polymerase chain reaction of 16S rDNA indicated lower levels of Pseudomonas aeruginosa and Porphyromonas gingivalis colonizing the dental plaques compared with the paired tracheal aspirate samples. These results support the hypothesis that dental bacteria may contribute to the pathology of severe AE-COPD. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Phylogeny and taxonomy of Echinodontium and related genera

Treesearch

Shi-Liang Liu; Yan Zhao; Yu-Cheng Dai; Karen K. Nakasone; Shuang-Hui He

2017-01-01

The phylogenetic relationship of eight species of Echinodontium, Laurilia, and Perplexostereum of Russulales were analyzed based on sequences of the nuc rDNA ITS1-5.8S-ITS2 (ITS [internal transcribed spacer]) and D1–D2 domains of nuc 28S rDNA (28S). Our results show that Echinodontium tinctorium, E. ryvardenii...

Mimosa caesalpiniifolia rhizobial isolates from different origins of the Brazilian Northeast.

PubMed

Martins, Paulo Geovani Silva; Junior, Mario Andrade Lira; Fracetto, Giselle Gomes Monteiro; da Silva, Maria Luiza Ribeiro Bastos; Vincentin, Rayssa Pereira; de Lyra, Maria do Carmo Catanho Pereira

2015-04-01

Biological nitrogen fixation from the legume-rhizobia symbiosis is one of the main sources of fixed nitrogen on land environments. Diazotrophic bacteria taxonomy has been substantially modified by the joint use of phenotypic, physiological and molecular aspects. Among these molecular tools, sequencing and genotyping of genomic regions such as 16S rDNA and repetitive conserved DNA regions have boosted the accuracy of species identification. This research is a phylogenetic study of diazotrophic bacteria from sabiá (Mimosa caesalpiniifolia Benth.), inoculated with soils from five municipalities of the Brazilian Northeast. After bacterial isolation and morphophysiological characterization, genotyping was performed using REP, ERIC and BOX oligonucleotides and 16S rDNA sequencing for genetic diversity identification. A 1.5b Kb fragment of the 16S rDNA was amplified from each isolate. Morphophysiological characterization of the 47 isolates created a dendrogram, where isolate PE-GR02 formed a monophyletic branch. The fingerprinting conducted with BOX, ERIC and REP shows distinct patterns, and their compilation created a dendrogram with diverse groups and, after blasting in GenBank, resulted in genetic identities ranging from 77 to 99 % with Burkholderia strains. The 16S rDNA phylogenetic tree constructed with these isolates and GenBank deposits of strains recommended for inoculant production confirm these isolates are distinct from the previously deposited strains, whereas isolates PE-CR02, PE-CR4, PE-CR07, PE-CR09 and PE-GE06 were the most distinct within the group. Morphophysiological characterization and BOX, ERIC and REP compilation enhanced the discrimination of the isolates, and the 16S rDNA sequences compared with GenBank confirmed the preference of Mimosa for Burkholderia diazotrophic bacteria.

Molecular approaches to differentiate three species of Nematodirus in sheep and goats from China based on internal transcribed spacer rDNA sequences.

PubMed

Zhao, G H; Jia, Y Q; Bian, Q Q; Nisbet, A J; Cheng, W Y; Liu, Y; Fang, Y Q; Ma, X T; Yu, S K

2015-05-01

Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.

Characterization of culturable anaerobic bacteria from the forestomach of an eastern grey kangaroo, Macropus giganteus.

PubMed

Ouwerkerk, D; Klieve, A V; Forster, R J; Templeton, J M; Maguire, A J

2005-01-01

To determine the culturable biodiversity of anaerobic bacteria isolated from the forestomach contents of an eastern grey kangaroo, Macropus giganteus, using phenotypic characterization and 16S rDNA sequence analysis. Bacteria from forestomach contents of an eastern grey kangaroo were isolated using anaerobic media containing milled curly Mitchell grass (Astrebla lappacea). DNA was extracted and the 16S rDNA sequenced for phylogenetic analysis. Forty bacterial isolates were obtained and placed in 17 groups based on phenotypic characteristics and restriction enzyme digestion of 16S rDNA PCR products. DNA sequencing revealed that the 17 groups comprised five known species (Clostridium butyricum, Streptococcus bovis, Clostridium sporogenes, Clostridium paraputrificum and Enterococcus avium) and 12 groups apparently representing new species, all within the phylum Firmicutes. Foregut contents from Australian macropod marsupials contain a microbial ecosystem with a novel bacterial biodiversity comprising a high percentage of previously unrecognized species. This study adds to knowledge of Australia's unique biodiversity, which may provide a future bioresource of genetic information and bacterial species of benefit to agriculture.

A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.

PubMed Central

Clos, J; Buttgereit, D; Grummt, I

1986-01-01

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I. Images PMID:3456157

[Phylogenetic relationships among the genera of Taxodiaceae and Cupressaceae from 28S rDNA sequences].

PubMed

Li, Chun-Xiang; Yang, Qun

2003-03-01

DNA sequences from 28S rDNA were used to assess relationships between and within traditional Taxodiaceae and Cupressaceae s.s. The MP tree and NJ tree generally are similar to one another. The results show that Taxodiaceae and Cupressaceae s.s. form a monophyletic conifer lineage excluding Sciadopitys. In the Taxodiaceae-Cupressaceae s.s. monophyletic group, the Taxodiaceae is paraphyletic. Taxodium, Glyptostrobus and Cryptomeria forming a clade(Taxodioideae), in which Glyptostrobus and Taxodium are closely related and sister to Cryptomeria; Sequoia, Sequoiadendron and Metasequoia are closely related to each other, forming another clade (Sequoioideae), in which Sequoia and Sequoiadendron are closely related and sister to Metasequoia; the seven genera of Cupressaceae s.s. are found to be closely related to form a monophyletic lineage (Cupressoideae). These results are basically similar to analyses from chloroplast gene data. But the relationships among Taiwania, Sequoioideae, Taxodioideae, and Cupressoideae remain unclear because of the slow evolution rate of 28S rDNA, which might best be answered by sequencing more rapidly evolving nuclear genes.

A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss.

PubMed

Andree, K B; MacConnell, E; Hedrick, R P

1998-10-08

A nested polymerase chain reaction (PCR) test was developed to amplify a segment of the 18S rRNA gene from Myxobolus cerebralis, the agent causing whirling disease in salmonid fish. The PCR amplifies a 415 bp amplicon that was identified by dideoxynucleotide terminated sequencing to be identical to the known 18S rDNA sequence of M. cerebralis. There was no amplification of genomic DNA from 4 other myxosporean parasites of salmonid fish from the genus Myxobolus including M. arcticus, M. insidiosus, M. neurobius, and M. squamalis. The efficacy of the PCR test to detect early infections was demonstrated by amplification of the 415 bp fragment from experimentally exposed rainbow trout Oncorhynchus mykiss at 2 h and at 1, 2, and 3 wk postexposure to actinosporean stages (triactinomyxons) of M. cerebralis. In contrast, standard microscopic examinations of stained tissue sections of the same fish used for PCR were less reliable in detecting the presence of the parasite. Additional examinations of fish 5 mo postexposure, after sporogenesis had occurred, found the PCR to be a more reliable indicator of infection than pepsin-trypsin digest (PTD) method, particularly when trout were experimentally exposed to low levels of the infectious stages of the parasite. The PCR was able to amplify to detectable levels the equivalent of a single sporoplasm of M. cerebralis as found in a tissue sample. This test improves the detection of M. cerebralis because it can detect the presence of the parasite: (1) in both hosts, (2) in all known stages of its life cycle, and (3) at lower thresholds than currently used diagnostic methods. Lastly, the PCR test is less susceptible to morphological misidentifications of the spores that can occur with current microscopic procedures.

PCR-based molecular discrimination of Pandora neoaphidis isolates from related entomopathogenic fungi and development of species-specific diagnostic primers.

PubMed

Tymon, Anna M; Shah, Paresh A; Pell, Judith K

2004-04-01

Studies were performed to assess the genetic variation amongst isolates of the aphid-pathogenic fungus Pandora neoaphidis (syn. Erynia neoaphidis). 37 isolates were examined, from a range of pest and non-pest aphid species, as well as 21 from eight other entomophthoralean species. Universal primers were used to amplify the ITS rDNA regions and all of the species tested produced discrete ITS groups, with the exception of Conidiobolus spp. Neighbour-joining analysis of the ITS2 regions from P. neoaphidis, P. kondoiensis and Zoophthora radicans demonstrated that these three species formed distinct groups with sequence identities of 58-82% between the groups. An ITS size of ca 1,100 bp was diagnostic for P. neoaphidis, while ca 1,450 bp was characteristic of P. kondoiensis. ITS-RFLP analysis failed to yield intraspecific polymorphisms in any of the P. neoaphidis isolates screened, although it was useful in distinguishing between different entomophthoralean species. Some intraspecific variation in the ITS region was detected in a number of isolates of Z. radicans and Conidiobolus spp. We propose that two isolates previously identified as P. neoaphidis based on conidia morphology, are actually P. kondoiensis based on molecular studies. Sequencing analysis of the complete ITS region from P. neoaphidis and P. kondoiensis allowed species-specific primers to be developed for P. neoaphidis and P. kondoiensis. These were used to screen aphids infected in laboratory bioassays and from field-collected samples, without prior isolation of the fungus. The primers are useful tools for quantifying the epizootiology of P. neoaphidis in aphid populations, as well as assessing competitive interactions between these two species.

5-Methyldeoxycytidine in the Physarum minichromosome containing the ribosomal RNA genes.

PubMed Central

Cooney, C A; Matthews, H R; Bradbury, E M

1984-01-01

5-Methyldeoxycytidine (5MC) was analyzed by high pressure liquid chromatography (HPLC) and by restriction enzyme digestion in rDNA isolated from Physarum polycephalum. rDNA from Physarum M3C strain microplasmodia has a significant 5MC content (about half that of the whole genomic DNA). This rDNA contains many C5MCGG sites because it is clearly digested further by Msp I than by Hpa II. However, most 5MC is in other sites. In particular, alternating CG sequences appear to be highly methylated. HPLC of deoxyribonucleosides shows tha most of the transcribed regions contain little or no 5MC. Restriction digestion indicates that there is little or no 5MC in any of the transcribed regions including the transcription origin and adjacent sequences. Over 90% of the total 5MC is in or near the central nontranscribed spacer and most methylated restriction sites are in inverted repeats of this spacer. rDNA is very heterogeneous with respect to 5MC. The 5MC pattern doesn't appear to change with inactivation of the rRNA genes during reversible differentiation from microplasmodia (growing) to microsclerotia (dormant), showing that inactivation is due to changes in other chromatin variables. The 5MC pattern is different between Physarum strains. The possible involvement of this 5MC in rDNA chromatin structure and in cruciform and Z-DNA formation is discussed. Images PMID:6322108

Molecular identification of arsenic-resistant estuarine bacteria and characterization of their ars genotype.

PubMed

Sri Lakshmi Sunita, M; Prashant, S; Bramha Chari, P V; Nageswara Rao, S; Balaravi, Padma; Kavi Kishor, P B

2012-01-01

In the present study, 44 arsenic-resistant bacteria were isolated through serial dilutions on agar plate with concentrations ≥0.05 mM of sodium arsenite and ≥10 mM of sodium arsenate from Mandovi and Zuari--estuarine water systems. The ars genotype characterization in 36 bacterial isolates (resistant to 100 mM of sodium arsenate) revealed that only 17 isolates harboured the arsA (ATPase), B (arsenite permease) and C (arsenate reductase) genes on the plasmid DNA. The arsA, B and C genes were individually detected using PCR in 16, 9 and 13 bacterial isolates respectively. Molecular identification of the 17 isolates bearing the ars genotype was carried using 16S rDNA sequencing. A 1300 bp full length arsB gene encoding arsenite efflux pump and a 409 bp fragment of arsC gene coding for arsenate reductase were isolated from the genera Halomonas and Acinetobacter. Phylogenetic analysis of arsB and arsC genes indicated their close genetic relationship with plasmid borne ars genes of E. coli and arsenate reductase of plant origin. The putative arsenate reductase gene isolated from Acinetobacter species complemented arsenate resistance in E. coli WC3110 and JM109 validating its function. This study dealing with isolation of native arsenic-resistant bacteria and characterization of their ars genes might be useful to develop efficient arsenic detoxification strategies for arsenic contaminated aquifers.

Human ribosomal RNA gene: nucleotide sequence of the transcription initiation region and comparison of three mammalian genes.

PubMed Central

Financsek, I; Mizumoto, K; Mishima, Y; Muramatsu, M

1982-01-01

The transcription initiation site of the human ribosomal RNA gene (rDNA) was located by using the single-strand specific nuclease protection method and by determining the first nucleotide of the in vitro capped 45S preribosomal RNA. The sequence of 1,211 nucleotides surrounding the initiation site was determined. The sequenced region was found to consist of 75% G and C and to contain a number of short direct and inverted repeats and palindromes. By comparison of the corresponding initiation regions of three mammalian species, several conserved sequences were found upstream and downstream from the transcription starting point. Two short A + T-rich sequences are present on human, mouse, and rat ribosomal RNA genes between the initiation site and 40 nucleotides upstream, and a C + T cluster is located at a position around -60. At and downstream from the initiation site, a common sequence, T-AG-C-T-G-A-C-A-C-G-C-T-G-T-C-C-T-CT-T, was found in the three genes from position -1 through +18. The strong conservation of these sequences suggests their functional significance in rDNA. The S1 nuclease protection experiments with cloned rDNA fragments indicated the presence in human 45S RNA of molecules several hundred nucleotides shorter than the supposed primary transcript. The first 19 nucleotides of these molecules appear identical--except for one mismatch--to the nucleotide sequence of the 5' end of a supposed early processing product of the mouse 45S RNA. Images PMID:6954460

Diversity of Cronobacter spp. isolates from the vegetables in the middle-east coastline of China.

PubMed

Chen, Wanyi; Yang, Jielin; You, Chunping; Liu, Zhenmin

2016-06-01

Cronobacter spp. has caused life-threatening neonatal infections mainly resulted from consumption of contaminated powdered infant formula. A total of 102 vegetable samples from retail markets were evaluated for the presence of Cronobacter spp. Thirty-five presumptive Cronobacter isolates were isolated and identified using API 20E and 16S rDNA sequencing analyses. All isolates and type strains were characterized using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and genetic profiles of cluster analysis from this molecular typing test clearly showed that there were differences among isolates from different vegetables. A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) based on the amplification of the gyrB gene (1258 bp) was developed to differentiate among Cronobacter species. A new PCR-RFLP assay based on the amplification of the gyrB gene using Alu I and Hinf I endonuclease combination is established and it has been confirmed an accurate and rapid subtyping method to differentiate Cronobacter species. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains, which has much better resolution based on SNPs in the identification of Cronobacter species specificity than PCR-RFLP and ERIC-PCR. Our study further confirmed that vegetables are one of the most common habitats or sources of Cronobacter spp. contamination in the middle-east coastline of China.

Phylogenetic analysis of two Plectus mitochondrial genomes (Nematoda: Plectida) supports a sister group relationship between Plectida and Rhabditida within Chromadorea.

PubMed

Kim, Jiyeon; Kern, Elizabeth; Kim, Taeho; Sim, Mikang; Kim, Jaebum; Kim, Yuseob; Park, Chungoo; Nadler, Steven A; Park, Joong-Ki

2017-02-01

Plectida is an important nematode order with species that occupy many different biological niches. The order includes free-living aquatic and soil-dwelling species, but its phylogenetic position has remained uncertain. We sequenced the complete mitochondrial genomes of two members of this order, Plectus acuminatus and Plectus aquatilis and compared them with those of other major nematode clades. The genome size and base composition of these species are similar to other nematodes; 14,831 and 14,372bp, respectively, with AT contents of 71.0% and 70.1%. Gene content was also similar to other nematodes, but gene order and coding direction of Plectus mtDNAs were dissimilar from other chromadorean species. P. acuminatus and P. aquatilis are the first chromadorean species found to contain a gene inversion. We reconstructed mitochondrial genome phylogenetic trees using nucleotide and amino acid datasets from 87 nematodes that represent major nematode clades, including the Plectus sequences. Trees from phylogenetic analyses using maximum likelihood and Bayesian methods depicted Plectida as the sister group to other sequenced chromadorean nematodes. This finding is consistent with several phylogenetic results based on SSU rDNA, but disagrees with a classification based on morphology. Mitogenomes representing other basal chromadorean groups (Araeolaimida, Monhysterida, Desmodorida, Chromadorida) are needed to confirm their phylogenetic relationships. Copyright © 2016 Elsevier Inc. All rights reserved.

FISH-aimed karyotyping and characterization of Renner complexes in permanent heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Joachimiak, Andrzej J

2005-02-01

Fluorescence in situ hybridization (FISH) using 25S rDNA, 5S rDNA, and telomere sequences as probes was carried out in the complex permanent heterozygote Rhoeo spathacea. Telomere sites were exclusively terminal. All 10 25S rDNA loci were located distally and appeared transcriptionally active after silver staining. Six distal and 2 interstitial 5S rDNA sites were detected; 2 of the distal sites strictly colocalized with 25S rDNA loci. The 2 intercalary 5S rDNA loci occurred in short arms of 2 chromosomes that conjoined at meiosis. Chromosomes differed as to the amount of AT-rich centric heterochromatin, suggesting involvement of pericentromeric regions in translocations. The possibility of Robertsonian-like rearrangements was discussed. Double target FISH with ribosomal probes along with DAPI fluorescence gave the basis for full chromosome identification in mitosis. The 2 Renner complexes are structurally balanced, both having 5 25S and 4 5S rDNA sites. Centromere clustering, telomere association, a high number of NOR sites, and a strong tendency for formation of joint nucleoli contribute to the preservation of highly polarized Rabl arrangement at interphase. These findings were discussed in relation to meiotic catenation in Rhoeo.

Sarocladium spinificis, a new endophytic species from the coastal grass Spinifex littoreus in Taiwan.

PubMed

Yeh, Yu-Hung; Kirschner, Roland

2014-12-01

Sarocladium species are frequently associated with grasses as saprobes, parasites, and mutualistic endophytes. A species of Sarocladium (anamorphic Hypocreales) was isolated as endophytic fungus from the coastal grass Spinifex littoreus (Poaceae). According to characterization by LSU and ITS rDNA sequences and culture morphology and micromorphology, the species differed from the species hitherto described in Sarocladium. A key to the known species of Sarocladium is given. Sarocladium spinificis is proposed as a new species. LSU rDNA sequences and conidiophore branching and conidium size are useful characters for distinguishing between species of Sarocladium.

5S ribosomal RNA database Y2K

PubMed Central

Szymanski, Maciej; Barciszewska, Miroslawa Z.; Barciszewski, Jan; Erdmann, Volker A.

2000-01-01

This paper presents the updated version (Y2K) of the database of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA), http://rose.man/poznan. pl/5SData/index.html . This edition of the database contains 1985 primary structures of 5S rRNA and 5S rDNA. They include 60 archaebacterial, 470 eubacterial, 63 plastid, nine mitochondrial and 1383 eukaryotic sequences. The nucleotide sequences of the 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of the source organisms. PMID:10592212

5S ribosomal RNA database Y2K.

PubMed

Szymanski, M; Barciszewska, M Z; Barciszewski, J; Erdmann, V A

2000-01-01

This paper presents the updated version (Y2K) of the database of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA), http://rose.man/poznan.pl/5SData/index.html. This edition of the database contains 1985primary structures of 5S rRNA and 5S rDNA. They include 60 archaebacterial, 470 eubacterial, 63 plastid, nine mitochondrial and 1383 eukaryotic sequences. The nucleotide sequences of the 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of the source organisms.

Taxonomy and phylogeny of Lopharia s.s., Dendrodontia, Dentocorticium and Fuscocerrena (Basidiomycota, Polyporales)

Treesearch

Shi-Liang Liu; Karen K. Nakasone; Sheng-Hua Wu; Shuang-Hui He; Yu-Cheng Dai

2018-01-01

Eleven taxa of Lopharia s.s., Dendrodontia, Dentocorticium and Fuscocerrena in Polyporales are included in the phylogenetic analyses of nuc rDNA ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (28S) and RNA polymerase II second-largest subunit (rpb2) sequences. New species Lopharia resupinata...

Bacterial endosymbioses of gutless tube-dwelling worms in nonhydrothermal vent habitats.

PubMed

Naganuma, Takeshi; Elsaied, Hosam E; Hoshii, Daiki; Kimura, Hiroyuki

2005-01-01

Gutless tube-dwelling worms of pogonophorans (also known as frenulates) and vestimentiferans depend on primary production of endosymbiotic bacteria. The endosymbionts include thiotrophs that oxidize sulfur for autotrophic production and methanotrophs that oxidize and assimilate methane. Although most of the pogonophoran and vestimentiferan tube worms possess single thiotrophic 16S rRNA genes (16S rDNA) related to gamma-proteobacteria, some pogonohorans are known to bear single methanotroph species or even dual symbionts of thiotrophs and methanotrophs. The vestimentiferan Lamellibrachia sp. L1 shows symbiotic 16S rDNA sequences of alpha-, beta-, gamma-, and epsilon-proteobacteria, varying among specimens, with RuBisCO form II gene (cbbM) sequences related to beta-proteobacteria. An unidentified pogonophoran from the world's deepest cold seep, 7326-m deep in the Japan Trench, hosts a symbiotic thiotroph based on 16S rDNA with the RuBisCO form I gene (cbbL). In contrast, a shallow-water pogonophoran (Oligobrachia mashikoi) in coastal Japan Sea has a methanotrophic 16S rDNA and thiotrophic cbbL, which may suggest the feature of type X methanotrophs. These observations demonstrate that pogonophoran and vestimentiferan worms have higher plasticity in bacterial symbioses than previously suspected.

The phylogeny of termites (Dictyoptera: Isoptera) based on mitochondrial and nuclear markers: Implications for the evolution of the worker and pseudergate castes, and foraging behaviors.

PubMed

Legendre, Frédéric; Whiting, Michael F; Bordereau, Christian; Cancello, Eliana M; Evans, Theodore A; Grandcolas, Philippe

2008-08-01

A phylogenetic hypothesis of termite relationships was inferred from DNA sequence data. Seven gene fragments (12S rDNA, 16S rDNA, 18S rDNA, 28S rDNA, cytochrome oxidase I, cytochrome oxidase II and cytochrome b) were sequenced for 40 termite exemplars, representing all termite families and 14 outgroups. Termites were found to be monophyletic with Mastotermes darwiniensis (Mastotermitidae) as sister group to the remainder of the termites. In this remainder, the family Kalotermitidae was sister group to other families. The families Kalotermitidae, Hodotermitidae and Termitidae were retrieved as monophyletic whereas the Termopsidae and Rhinotermitidae appeared paraphyletic. All of these results were very stable and supported with high bootstrap and Bremer values. The evolution of worker caste and foraging behavior were discussed according to the phylogenetic hypothesis. Our analyses suggested that both true workers and pseudergates ("false workers") were the result of at least two different origins. Our data support a traditional hypothesis of foraging behavior, in which the evolutionary transition from a one-piece type to a separate life type occurred through an intermediate behavioral form.

Molecular Systematic of Three Species of Oithona (Copepoda, Cyclopoida) from the Atlantic Ocean: Comparative Analysis Using 28S rDNA

PubMed Central

Cepeda, Georgina D.; Blanco-Bercial, Leocadio; Bucklin, Ann; Berón, Corina M.; Viñas, María D.

2012-01-01

Species of Oithona (Copepoda, Cyclopoida) are highly abundant, ecologically important, and widely distributed throughout the world oceans. Although there are valid and detailed descriptions of the species, routine species identifications remain challenging due to their small size, subtle morphological diagnostic traits, and the description of geographic forms or varieties. This study examined three species of Oithona (O. similis, O. atlantica and O. nana) occurring in the Argentine sector of the South Atlantic Ocean based on DNA sequence variation of a 575 base-pair region of 28S rDNA, with comparative analysis of these species from other North and South Atlantic regions. DNA sequence variation clearly resolved and discriminated the species, and revealed low levels of intraspecific variation among North and South Atlantic populations of each species. The 28S rDNA region was thus shown to provide an accurate and reliable means of identifying the species throughout the sampled domain. Analysis of 28S rDNA variation for additional species collected throughout the global ocean will be useful to accurately characterize biogeographical distributions of the species and to examine phylogenetic relationships among them. PMID:22558245

New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis.

PubMed

Smith, Michael G; Gianoulis, Tara A; Pukatzki, Stefan; Mekalanos, John J; Ornston, L Nicholas; Gerstein, Mark; Snyder, Michael

2007-03-01

Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicemia, and urinary tract infections. We explored the pathogenic content of this harmful pathogen using a combination of DNA sequencing and insertional mutagenesis. The genome of this organism was sequenced using a strategy involving high-density pyrosequencing, a novel, rapid method of high-throughput sequencing. Excluding the rDNA repeats, the assembled genome is 3,976,746 base pairs (bp) and has 3830 ORFs. A significant fraction of ORFs (17.2%) are located in 28 putative alien islands, indicating that the genome has acquired a large amount of foreign DNA. Consistent with its role in pathogenesis, a remarkable number of the islands (16) contain genes implicated in virulence, indicating the organism devotes a considerable portion of its genes to pathogenesis. The largest island contains elements homologous to the Legionella/Coxiella Type IV secretion apparatus. Type IV secretion systems have been demonstrated to be important for virulence in other organisms and thus are likely to help mediate pathogenesis of A. baumannii. Insertional mutagenesis generated avirulent isolates of A. baumannii and verified that six of the islands contain virulence genes, including two novel islands containing genes that lacked homology with others in the databases. The DNA sequencing approach described in this study allows the rapid elucidation of the DNA sequence of any microbe and, when combined with genetic screens, can identify many novel genes important for microbial pathogenesis.

Details of the evolutionary history from invertebrates to vertebrates, as deduced from the sequences of 18S rDNA.

PubMed Central

Wada, H; Satoh, N

1994-01-01

Almost the entire sequences of 18S rDNA were determined for two chaetognaths, five echinoderms, a hemichordate, and two urochordates (a larvacean and a salp). Phylogenetic comparisons of the sequences, together with those of other deuterostomes (an ascidian, a cephalochordate, and vertebrates) and protostomes (an arthropod and a mollusc), suggest the monophyly of the deuterostomes, with the exception of the chaetognaths. Chaetognaths may not be a group of deuterostomes. The deuterostome group closest to vertebrates was the group of cephalochordates. Ascidians, larvaceans, and salps seem to form a discrete group (urochordates), in which the early divergence of larvaceans is evident. These results support the hypothesis that chordates evolved from free-living ancestors. PMID:8127885

Evidence for 5S rDNA Horizontal Transfer in the toadfish Halobatrachus didactylus (Schneider, 1801) based on the analysis of three multigene families

PubMed Central

2012-01-01

Background The Batrachoididae family is a group of marine teleosts that includes several species with more complicated physiological characteristics, such as their excretory, reproductive, cardiovascular and respiratory systems. Previous studies of the 5S rDNA gene family carried out in four species from the Western Atlantic showed two types of this gene in two species but only one in the other two, under processes of concerted evolution and birth-and-death evolution with purifying selection. Here we present results of the 5S rDNA and another two gene families in Halobatrachus didactylus, an Eastern Atlantic species, and draw evolutionary inferences regarding the gene families. In addition we have also mapped the genes on the chromosomes by two-colour fluorescence in situ hybridization (FISH). Results Two types of 5S rDNA were observed, named type α and type β. Molecular analysis of the 5S rDNA indicates that H. didactylus does not share the non-transcribed spacer (NTS) sequences with four other species of the family; therefore, it must have evolved in isolation. Amplification with the type β specific primers amplified a specific band in 9 specimens of H. didactylus and two of Sparus aurata. Both types showed regulatory regions and a secondary structure which mark them as functional genes. However, the U2 snRNA gene and the ITS-1 sequence showed one electrophoretic band and with one type of sequence. The U2 snRNA sequence was the most variable of the three multigene families studied. Results from two-colour FISH showed no co-localization of the gene coding from three multigene families and provided the first map of the chromosomes of the species. Conclusions A highly significant finding was observed in the analysis of the 5S rDNA, since two such distant species as H. didactylus and Sparus aurata share a 5S rDNA type. This 5S rDNA type has been detected in other species belonging to the Batrachoidiformes and Perciformes orders, but not in the Pleuronectiformes and Clupeiformes orders. Two hypotheses have been outlined: one is the possible vertical permanence of the shared type in some fish lineages, and the other is the possibility of a horizontal transference event between ancient species of the Perciformes and Batrachoidiformes orders. This finding opens a new perspective in fish evolution and in the knowledge of the dynamism of the 5S rDNA. Cytogenetic analysis allowed some evolutionary trends to be roughed out, such as the progressive change in the U2 snDNA and the organization of (GATA)n repeats, from dispersed to localized in one locus. The accumulation of (GATA)n repeats in one chromosome pair could be implicated in the evolution of a pair of proto-sex chromosomes. This possibility could situate H. didactylus as the most highly evolved of the Batrachoididae family in terms of sex chromosome biology. PMID:23039906

Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

PubMed Central

Maggert, Keith A.

2014-01-01

The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings.

PubMed

Schabereiter-Gurtner, C; Piñar, G; Vybiral, D; Lubitz, W; Rölleke, S

2001-11-01

A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.

Identification and chromosome mapping of repetitive elements in the Astyanax scabripinnis (Teleostei: Characidae) species complex.

PubMed

Barbosa, Patrícia; de Oliveira, Luiz Antonio; Pucci, Marcela Baer; Santos, Mateus Henrique; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo; Nogaroto, Viviane; de Almeida, Mara Cristina; Artoni, Roberto Ferreira

2015-02-01

Most part of the eukaryotic genome is composed of repeated sequences or multiple copies of DNA, which were considered as "junk DNA", and may be associated to the heterochromatin. In this study, three populations of Astyanax aff. scabripinnis from Brazilian rivers of Guaratinguetá and Pindamonhangaba (São Paulo) and a population from Maringá (Paraná) were analyzed concerning the localization of the nucleolar organizer regions (Ag-NORs), the As51 satellite DNA, the 18S ribosomal DNA (rDNA), and the 5S rDNA. Repeated sequences were also isolated and identified by the Cot - 1 method, which indicated similarity (90%) with the LINE UnaL2 retrotransposon. The fluorescence in situ hybridization (FISH) showed the retrotransposon dispersed and more concentrated markers in centromeric and telomeric chromosomal regions. These sequences were co-localized and interspaced with 18S and 5S rDNA and As51, confirmed by fiber-FISH essay. The B chromosome found in these populations pointed to a conspicuous hybridization with LINE probe, which is also co-located in As51 sequences. The NORs were active at unique sites of a homologous pair in the three populations. There were no evidences that transposable elements and repetitive DNA had influence in the transcriptional regulation of ribosomal genes in our analyses.

Redescription and Molecular Assessment of Relationships Among Three Species of Echeneibothrium (Rhinebothriidea: Echeneibothriidae) Parasitizing the Yellownose Skate, Dipturus chilensis, in Chile.

PubMed

Bueno, Veronica M; Caira, Janine N

2017-06-01

Much progress has recently been made in revising the taxonomic assignments of genera originally classified in the polyphyletic "Tetraphyllidea." Many of these genera, including Echeneibothrium, were accommodated in the order Rhinebothriidea. However, beyond this larger taxonomic action, little work has been conducted on this genus over the past 50 yr. Consequently, the criteria used for characterizing species of Echeneibothrium have lagged behind those typically used in more modern descriptions of elasmobranch-hosted cestode taxa. A series of collecting trips to Chile to obtain cestodes from the yellownose skate, Dipturus chilensis , provided a unique opportunity to apply modern morphological and molecular methods to investigate the 3 species of Echeneibothrium reported parasitizing this skate, specifically Echeneibothrium megalosoma, Echeneibothrium multiloculatum, and Echeneibothrium williamsi. In addition to redescribing all 3 species, using morphological data from light and scanning electron microscopy, maximum likelihood and bayesian inference phylogenetic analyses of the D1-D3 regions of the 28S rDNA gene were conducted to assess their relationships among other echeneibothriids for which comparable data are available. Sequencing of 59 specimens representing these 3 species of Echeneibothrium allowed us to assess the intra- and interspecific variation in the 28S rDNA gene. The redescriptions use standardized terminology for scolex morphology, proglottid anatomy, and microthrix forms and pattern; they also expand on the original descriptions to include data on scolex size, ovary size, vas deferens and vaginal configurations, testes arrangement, and genital pore position. Our morphological work led to a major reinterpretation of the scolex morphology with the recognition that all 3 species bear an apical bothridial sucker, rather than an apical loculus, prompting emendation of the diagnosis for the family Echeneibothriidae. The presence of a band of spinitriches at the apex of the apical modification of the scolex proper seems to represent an important feature for distinguishing the 2 portions of the myzorhynchus across species. Intraspecific variation ranged from 0 to 7 bp across species and interspecific variation ranged from a low of 39-46 bp between E. williamsi and E. multiloculatum to a high of 61-66 bp between E. multiloculatum and E. megalosoma. Phylogenetic analyses indicate that the 3 species of Echeneibothrium hosted by the yellownose skate are not each other's closest relatives, suggesting multiple colonization events of D. chilensis have occurred. Further phylogenetic investigation is also likely to confirm the status of the genus Pseudanthobothrium as a synonym of Echeneibothrium because its species generally group among members of Echeneibothrium.

Morphology and molecular characterization of the epiphytic dinoflagellate Prorocentrum cf. rhathymum in temperate waters off Jeju Island, Korea

NASA Astrophysics Data System (ADS)

Lim, An Suk; Jeong, Hae Jin; Jang, Tae Young; Kang, Nam Seon; Lee, Sung Yeon; Yoo, Yeong Du; Kim, Hyung Seop

2013-03-01

Prorocentrum spp. are planktonic and/or benthic species. Benthic Prorocentrum species are of primary concern to scientists and the public because some of them are toxic. We established clonal cultures of 3 strains of Prorocentrum species that were collected from the thalli of a macroalga in the coastal waters off Jeju Island, located at the southern end of Korea. The Korean strains of P. cf. rhathymum, which are morphologically almost identical to the Virgin Island strain of P. rhathymum, were different from P. mexicanum because the former dinoflagellate has one simple collar-like spine in the periflagellar area, while the latter dinoflagellate has a 2- or 3-horned spine. In addition, the sequences of the small subunit (SSU) rDNA of the Korean strains were identical to those of the Malaysian and Floridian strains of P. rhathymum, while the sequences of the large subunit (LSU) rDNA of the Korean strains were 0.1-0.9% different from those of the Iranian and Malaysian strains of P. rhathymum. In phylogenetic trees based on the SSU rDNA sequences, the Korean strains of P. rhathymum formed a clade with the Malaysian and Floridian strains of P. rhathymum and the Vietnamese and Polynesian strains of P. mexicanum. However, in phylogenetic trees based on the LSU rDNA sequences, the Korean strains of P. rhathymum formed a clade with the Iranian strain of P. rhathymum and the Spanish and Mexican strains of P. mexicanum. Therefore, the molecular characterization of the Korean strains does not allow us to clearly classify them as P. rhathymum, nor P. mexicanum, although their morphology has so far been reported to be closer to that of P. rhathymum than P. mexicanum and thus we designated them as P. cf. rhathymum.

Cospeciation of Psyllids and Their Primary Prokaryotic Endosymbionts

PubMed Central

Thao, MyLo L.; Moran, Nancy A.; Abbot, Patrick; Brennan, Eric B.; Burckhardt, Daniel H.; Baumann, Paul

2000-01-01

Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity. Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies. In addition, 0.54-kb fragments of the psyllid nuclear gene wingless were also amplified from 26 species. Phylogenetic trees derived from 16S-23S rDNA and from the host wingless gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies. This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont. In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology. The 3′ end of the 16S rDNA of the P endosymbionts differs from that of other members of the domain Bacteria in the lack of a sequence complementary to the mRNA ribosome binding site. The rate of sequence change in the 16S-23S rDNA of the psyllid P endosymbiont was considerably higher than that of other bacteria, including other fast-evolving insect endosymbionts. The lineage consisting of the P endosymbionts of psyllids was given the designation Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.). PMID:10877784

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

PubMed

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-10-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

PubMed Central

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P.H.

2014-01-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. PMID:24609384

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture.

PubMed

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P H

2014-05-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes.

PubMed

Bierhoff, H; Schmitz, K; Maass, F; Ye, J; Grummt, I

2010-01-01

Alternative transcription of the same gene in sense and antisense orientation regulates expression of protein-coding genes. Here we show that noncoding RNA (ncRNA) in sense and antisense orientation also controls transcription of rRNA genes (rDNA). rDNA exists in two types of chromatin--a euchromatic conformation that is permissive to transcription and a heterochromatic conformation that is transcriptionally silent. Silencing of rDNA is mediated by NoRC, a chromatin-remodeling complex that triggers heterochromatin formation. NoRC function requires RNA that is complementary to the rDNA promoter (pRNA). pRNA forms a DNA:RNA triplex with a regulatory element in the rDNA promoter, and this triplex structure is recognized by DNMT3b. The results imply that triplex-mediated targeting of DNMT3b to specific sequences may be a common pathway in epigenetic regulation. We also show that rDNA is transcribed in antisense orientation. The level of antisense RNA (asRNA) is down-regulated in cancer cells and up-regulated in senescent cells. Ectopic asRNA triggers trimethylation of histone H4 at lysine 20 (H4K20me3), suggesting that antisense transcripts guide the histone methyltransferase Suv4-20 to rDNA. The results reveal that noncoding RNAs in sense and antisense orientation are important determinants of the epigenetic state of rDNA.

Revisiting the phylogeny of Ocellularieae, the second largest tribe within Graphidaceae (lichenized Ascomycota: Ostropales)

Treesearch

Ekaphan Kraichak; Sittiporn Parnmen; Robert Lücking; Eimy Rivas Plata; Andre Aptroot; Marcela E.S. Caceres; Damien Ertz; Armin Mangold; Joel A. Mercado-Diaz; Khwanruan Papong; Dries Van der Broeck; Gothamie Weerakoon; H. Thorsten Lumbsch; NO-VALUE

2014-01-01

We present an updated 3-locus molecular phylogeny of tribe Ocellularieae, the second largest tribe within subfamily Graphidoideae in the Graphidaceae. Adding 165 newly generated sequences from the mitochondrial small subunit rDNA (mtSSU), the nuclear large subunit rDNA (nuLSU), and the second largest subunit of the DNA-directed RNA polymerase II (RPB2), we currently...

Myxobolus cerebralis internal transcribed spacer 1 (ITS-1) sequences support recent spread of the parasite to North America and within Europe

USGS Publications Warehouse

Whipps, Christopher M.; El-Matbouli, M.; Hedrick, R.P.; Blazer, V.; Kent, M.L.

2004-01-01

Molecular approaches for resolving relationships among the Myxozoa have relied mainly on small subunit (SSU) ribosomal DNA (rDNA) sequence analysis. This region of the gene is generally used for higher phylogenetic studies, and the conservative nature of this gene may make it inadequate for intraspecific comparisons. Previous intraspecific studies of Myxobolus cerebralis based on molecular analyses reported that the sequence of SSU rDNA and the internal transcribed spacer (ITS) were highly conserved in representatives of the parasite from North America and Europe. Considering that the ITS is usually a more variable region than the SSU, we reanalyzed available sequences on GenBank and obtained sequences from other M. cerebralis representatives from the states of California and West Virginia in the USA and from Germany and Russia. With the exception of 7 base pairs, most of the sequence designated as ITS-1 in GenBank was a highly conserved portion of the rDNA near the 3-prime end of the SSU region. Nonetheless, the additional ITS-1 sequences obtained from the available geographic representatives were well conserved. It is unlikely that we would have observed virtually identical ITS-1 sequences between European and American M. cerebralis samples had it spread naturally over time, particularly when compared to the variation seen between isolates of another myxozoan (Kudoa thyrsites) that has most likely spread naturally. These data further support the hypothesis that the current distribution of M. cerebralis in North America is a result of recent introductions followed by dispersal via anthropogenic means, largely through the stocking of infected trout for sport fishing.

Ribosomal DNA Organization Before and After Magnification in Drosophila melanogaster

PubMed Central

Bianciardi, Alessio; Boschi, Manuela; Swanson, Ellen E.; Belloni, Massimo; Robbins, Leonard G.

2012-01-01

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb− chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb2 mutant and in some magnified bb+ alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb2 allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids. PMID:22505623

Evolution of helotialean fungi (Leotiomycetes, Pezizomycotina): a nuclear rDNA phylogeny.

PubMed

Wang, Zheng; Binder, Manfred; Schoch, Conrad L; Johnston, Peter R; Spatafora, Joseph W; Hibbett, David S

2006-11-01

The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classification and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classification. Data sets with different compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Leotiomycetes, including the Helotiales, Erysiphales, Cyttariales, Rhytismatales, and Myxotrichaceae, is monophyletic; and (4) nine clades can be recognized within the Helotiales.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

PubMed Central

Bishop, R.P.; Hemmink, J.D.; Morrison, W.I.; Weir, W.; Toye, P.G.; Sitt, T.; Spooner, P.R.; Musoke, A.J.; Skilton, R.A.; Odongo, D.O.

2015-01-01

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. ‘Deep 454 pyrosequencing’ of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva. PMID:26543804

Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria

PubMed Central

2008-01-01

Background Polypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction (LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa. Results Analyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction. Conclusion By including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa. PMID:18471296

Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria.

PubMed

Evans, Nathaniel M; Lindner, Alberto; Raikova, Ekaterina V; Collins, Allen G; Cartwright, Paulyn

2008-05-09

Polypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction (LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa. Analyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction. By including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa.

Molecular cloning and sequence analysis of full-length growth hormone cDNAs from six important economic fishes.

PubMed

Zhang, Jing-Nan; Song, Ping; Hu, Jia-Rui; Mo, Sai-Jun; Peng, Mao-Yu; Zhou, Wei; Zou, Ji-Xing; Hu, Yin-Chang

2005-01-01

In this study,the full-length cDNAs of GH (Growth Hormone) gene was isolated from six important economic fishes, Siniperca kneri, Epinephelus coioides, Monopterus albus, Silurus asotus, Misgurnus anguillicaudatus and Carassius auratus gibelio Bloch. It is the first time to clone these GH sequences except E. coioides GH. The lengths of the above cDNAs are as follows: 953 bp, 1 023 bp, 825 bp, 1 082 bp, 1 154 bp and 1 180 bp. Each sequence includes an ORF of about 600 bp which encodes a protein of about 200 amino acid: S. kneri, E. coioides and M. albus GHs of 204 amino acid, S. asotus GH of 200 amino acid, M. anguillicaudatus and C. auratus gibelio GHs of 210 amino acid. Then detailed sequence analysis of the six GHs with many other fish sequences was performed. The six sequences all showed high homology to other sequences, especially to sequences within the same order, and many conserved residues were identified, most localized in five domains. The phylogenetic trees (MP and NJ) of many fish GH ORF sequences (including the new six) with Amia calva as outgroup were generally resolved and largely congruent with the morphology-based tree though some incongruities were observed, suggesting GH ORF should be paid more attention to in teleostean phylogeny.

Molecular characterization of the Indian poultry nodular tapeworm, Raillietina echinobothrida (Cestoda: Cyclophyllidea: Davaineidae) based on rDNA internal transcribed spacer 2 region.

PubMed

Ramnath; Jyrwa, D B; Dutta, A K; Das, B; Tandon, V

2014-03-01

The nodular tapeworm, Raillietina echinobothrida is a well studied avian gastrointestinal parasite of family Davaineidae (Cestoda: Cyclophyllidea). It is reported to be the largest in size and second most prevalent species infecting chicken in north-east India. In the present study, morphometrical methods coupled with the molecular analysis of the second internal transcribed spacer (ITS2) region of ribosomal DNA were employed for precise identification of the parasite. The annotated ITS2 region was found to be 446 bp long and further utilized to elucidate the phylogenetic relationships and its species-interrelationships at the molecular level. In phylogenetic analysis similar topology was observed among the trees obtained by distance-based neighbor-joining as well as character-based maximum parsimony tree building methods. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all Raillietina species well separated from the other cyclophyllidean (taeniid and hymenolepid) cestodes, while Diphyllobothrium latum (Pseudophyllidea: Diphyllobothriidae) was rooted as an out-group. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of order Cyclophyllidea against the out-group order Pseudophyllidea. The present study strengthens the potential of ITS2 as a reliable marker for phylogenetic reconstructions.

Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

PubMed Central

Gibbons, John G.; Branco, Alan T.; Godinho, Susana A.; Yu, Shoukai; Lemos, Bernardo

2015-01-01

Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome. PMID:25583482

[Phylogenetic relationships among members of the subfamily sedoideae (Crassulaceae) inferred from the ITS region sequences of nuclear rDNA].

PubMed

Goncharova, S B; Artiukova, E V; Goncharov, A A

2006-06-01

Nucleotide sequences of the nuclear rDNA ITS regions were determined in 20 species of the subfamily Sedoideae (Crassulaceae). The phylogenetic relationships of these species with other members of the subfamily, occurring mainly in Southeast Asia, were analyzed. It was shown that the genus Orostachys was not monophyletic; its typical subsection was reliably included into the clade of the genus Hylotelephium. Synapomorphic substitutions and indels, specific for the subsection Orostachys, were detected in ITS1. Sister relationships were established between clades Aizopsis and Phedimus, based on which they can be recognized as isolated genera.

Culturable bacteria present in the fluid of the hooded-pitcher plant Sarracenia minor based on 16S rDNA gene sequence data.

PubMed

Siragusa, Alex J; Swenson, Janice E; Casamatta, Dale A

2007-08-01

The culturable microbial community within the pitcher fluid of 93 Sarracenia minor carnivorous plants was examined over a 2-year study. Many aspects of the plant/bacterial/insect interaction within the pitcher fluid are minimally understood because the bacterial taxa present in these pitchers have not been identified. Thirteen isolates were characterized by 16S rDNA sequencing and subsequent phylogenetic analysis. The Proteobacteria were the most abundant taxa and included representatives from Serratia, Achromobacter, and Pantoea. The Actinobacteria Micrococcus was also abundant while Bacillus, Lactococcus, Chryseobacterium, and Rhodococcus were infrequently encountered. Several isolates conformed to species identifiers (>98% rDNA gene sequence similarity) including Serratia marcescens (isolates found in 27.5% of pitchers), Achromobacter xylosoxidans (37.6%), Micrococcus luteus (40.9%), Bacillus cereus (isolates found in 10.2%), Bacillus thuringiensis (5.4%), Lactococcus lactis (17.2%), and Rhodococcus equi (2.2%). Species-area curves suggest that sampling efforts were sufficient to recover a representative culturable bacterial community. The bacteria present represent a diverse community probably as a result of introduction by insect vectors, but the ecological significance remains under explored.

[Isolation and characterization of a new glyphosate-resistant strain from extremely polluted environment].

PubMed

Sh, Jiying; Jin, Dan; Lu, Wei; Zhang, Xiaoyu; Zhang, Chao; Li, Liang; Ma, Ruiqiang; Xiao, Lei; Wang, Yiding; Lin, Min

2008-06-01

To isolate and characterize a glyphosate-resistant strain from extremely polluted environment. A glyphosate-resistant strain was isolated from extremely polluted soil taking glyphosate as the selection pressure. Its glyphosate resistance, growth optimal pH and antibiotic sensitivity were detected. Its morphology, cultural characteristics, physiological and biochemical properties, chemotaxonomy and 16S rDNA sequences were studied. Based on these results, the strain was identified according to the ninth edition of Bergey's manual of determinative bacteriology. The isolate was named SL06500. It could grow in M9 minimal medium containing up to 500 mmol/L glyphosate. The cell growth optimal pH of SL06500 was 4.0. It was resistant to ampicillin, kanamycin, tetracycline and chloromycetin. The 16S rDNA of SL06500 was amplified by PCR and sequenced. Compared with the published nucleotide sequence of 16S rDNA in NCBI (National Center for Biotechnology Information), SL06500 showed high identity with Achromobacter and Alcaligenes. Based on morphological, physiological and biochemical characteristics, the strain was identified as Alcaligenes xylosoxidans subsp.xylosoxidans SL06500 according to the ninth edition of Bergey's manual of determinative bacteriology. Strain SL06500 is worthy to be studied because of its high glyphosate resistance.

Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses.

PubMed

Lee, I M; Bartoszyk, I M; Gundersen-Rindal, D E; Davis, R E

1997-07-01

A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera.

Marine Fungi: Their Ecology and Molecular Diversity

NASA Astrophysics Data System (ADS)

Richards, Thomas A.; Jones, Meredith D. M.; Leonard, Guy; Bass, David

2012-01-01

Fungi appear to be rare in marine environments. There are relatively few marine isolates in culture, and fungal small subunit ribosomal DNA (SSU rDNA) sequences are rarely recovered in marine clone library experiments (i.e., culture-independent sequence surveys of eukaryotic microbial diversity from environmental DNA samples). To explore the diversity of marine fungi, we took a broad selection of SSU rDNA data sets and calculated a summary phylogeny. Bringing these data together identified a diverse collection of marine fungi, including sequences branching close to chytrids (flagellated fungi), filamentous hypha-forming fungi, and multicellular fungi. However, the majority of the sequences branched with ascomycete and basidiomycete yeasts. We discuss evidence for 36 novel marine lineages, the majority and most divergent of which branch with the chytrids. We then investigate what these data mean for the evolutionary history of the Fungi and specifically marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine ecosystems.

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris).

PubMed

Zhang, Wenping; Zhang, Zhihe; Shen, Fujun; Hou, Rong; Lv, Xiaoping; Yue, Bisong

2006-08-01

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) of Panthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8-17 million years ago in the tiger and 4.6-16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular 'fossils' that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.

Candida adriatica sp. nov. and Candida molendinolei sp. nov., two yeast species isolated from olive oil and its by-products.

PubMed

Čadež, Neža; Raspor, Peter; Turchetti, Benedetta; Cardinali, Gianluigi; Ciafardini, Gino; Veneziani, Gianluca; Péter, Gábor

2012-09-01

Thirteen strains isolated from virgin olive oil or its by-products in several Mediterranean countries were found to be phenotypically and genetically divergent from currently recognized yeast species. Sequence analysis of the large subunit (LSU) rDNA D1/D2 domain and internal transcribed spacer regions/5.8S rDNA revealed that the strains represented two novel species described as Candida adriatica sp. nov. (type strain ZIM 2334(T) = CBS 12504(T) = NCAIM Y.02001(T)) and Candida molendinolei sp. nov. (type strain DBVPG 5508(T) = CBS 12508(T) = NCAIM Y.02000(T)). Phylogenetic analysis based on concatenated sequences of the small subunit rRNA gene, the D1/D2 region of the LSU rDNA and the translation elongation factor-1α gene suggested that C. adriatica sp. nov. and C. molendinolei sp. nov. should be placed within the Lindnera and Nakazawaea clades, respectively.

Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana.

PubMed

Escalante-Minakata, P; Blaschek, H P; Barba de la Rosa, A P; Santos, L; De León-Rodríguez, A

2008-06-01

To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana. The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro-organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae, Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria, Weissella paramesenteroides, Lactobacillus pontis, Lactobacillus kefiri, Lactobacillus plantarum and Lactobacillus farraginis. The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis. Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal. We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.

Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

PubMed

Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

2007-08-01

The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control.

Isolation and identification of efficient Egyptian malathion-degrading bacterial isolates.

PubMed

Hamouda, S A; Marzouk, M A; Abbassy, M A; Abd-El-Haleem, D A; Shamseldin, Abdelaal

2015-03-01

Bacterial isolates degrading malathion were isolated from the soil and agricultural waste water due to their ability to grow on minimal salt media amended with malathion as a sole carbon source. Efficiencies of native Egyptian bacterial malathion-degrading isolates were investigated and the study generated nine highly effective malathion-degrading bacterial strains among 40. Strains were identified by partial sequencing of 16S rDNA analysis. Comparative analysis of 16S rDNA sequences revealed that these bacteria are similar with the genus Acinetobacter and Bacillus spp. and RFLP based PCR of 16S rDNA gave four different RFLP patterns among strains with enzyme HinfI while with enzyme HaeI they gave two RFLP profiles. The degradation rate of malathion in liquid culture was estimated using gas chromatography. Bacterial strains could degrade more than 90% of the initial malathion concentration (1000 ppm) within 4 days. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Innovative Methods for Integrating Knowledge for Long-Term Monitoring of Contaminated Groundwater Sites: Understanding Microorganism Communities and their Associated Hydrochemical Environment

NASA Astrophysics Data System (ADS)

Mouser, P. J.; Rizzo, D. M.; Druschel, G.; O'Grady, P.; Stevens, L.

2005-12-01

This interdisciplinary study integrates hydrochemical and genome-based data to estimate the redox processes occurring at long-term monitoring sites. Groundwater samples have been collected from a well-characterized landfill-leachate contaminated aquifer in northeastern New York. Primers from the 16S rDNA gene were used to amplify Bacteria and Archaea in groundwater taken from monitoring wells located in clean, fringe, and contaminated locations within the aquifer. PCR-amplified rDNA were digested with restriction enzymes to evaluate terminal restriction fragment length polymorphism (T-RFLP) community profiles. The rDNA was cloned, sequenced, and partial sequences were matched against known organisms using the NCBI Blast database. Phylogenetic trees and bootstrapping were used to identify classifications of organisms and compare the communities from clean, fringe, and contaminated locations. We used Artificial Neural Network (ANN) models to incorporate microbial data with hydrochemical information for improving our understanding of subsurface processes.

Complete Genome Sequence of Methylobacterium populi P-1M, Isolated from Pink-Pigmented Household Biofilm

PubMed Central

Morohoshi, Tomohiro

2016-01-01

Methylobacterium populi P-1M is isolated from the pink-pigmented household biofilm. Here, we present the complete genome sequence of P-1M, consisting of one chromosome of 5,705,640 bp and five plasmids of 64,864 bp, 59,879 bp, 42,569 bp, 41,417 bp, and 29,506 bp. PMID:27313289

Strawberry disease lesions in rainbow trout from southern Idaho are associated with DNA from a Rickettsia-like organism.

PubMed

Lloyd, Sonja J; LaPatra, Scott E; Snekvik, Kevin R; St-Hilaire, Sophie; Cain, Kenneth D; Call, Douglas R

2008-11-20

Strawberry disease (SD) in the USA is a skin disorder of unknown etiology that occurs in rainbow trout Oncorhynchus mykiss and is characterized by bright red inflammatory lesions. To identify a candidate bacterial agent responsible for SD, we constructed 16S rDNA libraries from 7 SD lesion samples and 2 apparently healthy skin samples from SD-affected fish. A 16S rDNA sequence highly similar to members of the order Rickettsiales was present in 3 lesion libraries at 1%, 32% and 54% prevalence, but this sequence was not found in either healthy tissue library. Based on phylogenetic analysis, this Rickettsia-like organism (RLO) sequence is most closely related to 16S rDNA sequences of bacteria that may form a novel lineage within the Rickettsiales. We used nested PCR assays to screen 25 SD-affected fish for RLO or Flavobacterium psychrophilum DNA. Sixteen lesion samples were positive for the RLO sequence and 4 of the matched healthy samples were positive resulting in a significant association between SD lesions and presence of RLO DNA. While F. psychrophilum is reportedly associated with 'cold water strawberry disease' in the UK, we found no significant association between SD lesions and the presence of F. psychrophilum DNA. The statistical association between SD lesions and presence of RLO DNA is not proof of etiology, but these data suggest that RLO may play a role in SD in southern Idaho, USA.

The rDNA ITS region in the lessepsian marine angiosperm Halophila stipulacea (Forssk.) Aschers. (Hydrocharitaceae): intragenomic variability and putative pseudogenic sequences.

PubMed

Ruggiero, Maria Valeria; Procaccini, Gabriele

2004-01-01

Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea. This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin. In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis. A high degree of intraindividual variability of ITS sequences was found. Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals. Features of ITS functional sequences and pseudogenes are described. Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.

Organization and variation analysis of 5S rDNA in gynogenetic offspring of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂).

PubMed

Qin, QinBo; Wang, Juan; Wang, YuDe; Liu, Yun; Liu, ShaoJun

2015-03-13

The offspring with 100 chromosomes (abbreviated as GRCC) have been obtained in the first generation of Carassius auratus red var. (abbreviated as RCC, 2n = 100) (♀) × Megalobrama amblycephala (abbreviated as BSB, 2n = 48) (♂), in which the females and unexpected males both are found. Chromosomal and karyotypic analysis has been reported in GRCC which gynogenesis origin has been suggested, but lack genetic evidence. Fluorescence in situ hybridization with species-specific centromere probes directly proves that GRCC possess two sets of RCC-derived chromosomes. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (abbreviated as NTS) reveals that three types of 5S rDNA class (class I; class II and class III) in GRCC are completely inherited from their female parent (RCC), and show obvious base variations and insertions-deletions. Fluorescence in situ hybridization with the entire 5S rDNA probe reveals obvious chromosomal loci (class I and class II) variation in GRCC. This paper provides directly genetic evidence that GRCC is gynogenesis origin. In addition, our result is also reveals that distant hybridization inducing gynogenesis can lead to sequence and partial chromosomal loci of 5S rDNA gene obvious variation.

Evolutionary insight on localization of 18S, 28S rDNA genes on homologous chromosomes in Primates genomes

PubMed Central

Mazzoleni, Sofia; Rovatsos, Michail; Schillaci, Odessa; Dumas, Francesca

2018-01-01

Abstract We explored the topology of 18S and 28S rDNA units by fluorescence in situ hybridization (FISH) in the karyotypes of thirteen species representatives from major groups of Primates and Tupaia minor (Günther, 1876) (Scandentia), in order to expand our knowledge of Primate genome reshuffling and to identify the possible dispersion mechanisms of rDNA sequences. We documented that rDNA probe signals were identified on one to six pairs of chromosomes, both acrocentric and metacentric ones. In addition, we examined the potential homology of chromosomes bearing rDNA genes across different species and in a wide phylogenetic perspective, based on the DAPI-inverted pattern and their synteny to human. Our analysis revealed an extensive variability in the topology of the rDNA signals across studied species. In some cases, closely related species show signals on homologous chromosomes, thus representing synapomorphies, while in other cases, signal was detected on distinct chromosomes, leading to species specific patterns. These results led us to support the hypothesis that different mechanisms are responsible for the distribution of the ribosomal DNA cluster in Primates. PMID:29416829

Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima

PubMed Central

Boutte, Julien; Aliaga, Benoît; Lima, Oscar; Ferreira de Carvalho, Julie; Ainouche, Abdelkader; Macas, Jiri; Rousseau-Gueutin, Mathieu; Coriton, Olivier; Ainouche, Malika; Salmon, Armel

2015-01-01

Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5′-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies. PMID:26530424

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

PubMed

Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Genetic variability in isolates of Chromobacterium violaceum from pulmonary secretion, water, and soil.

PubMed

Santini, A C; Magalhães, J T; Cascardo, J C M; Corrêa, R X

2016-04-28

Chromobacterium violaceum is a free-living Gram-negative bacillus usually found in the water and soil in tropical regions, which causes infections in humans. Chromobacteriosis is characterized by rapid dissemination and high mortality. The aim of this study was to detect the genetic variability among C. violaceum type strain ATCC 12472, and seven isolates from the environment and one from a pulmonary secretion from a chromobacteriosis patient from Ilhéus, Bahia. The molecular characterization of all samples was performed by polymerase chain reaction (PCR) sequencing and 16S rDNA analysis. Primers specific for two ATCC 12472 pathogenicity genes, hilA and yscD, as well as random amplified polymorphic DNA (RAPD), were used for PCR amplification and comparative sequencing of the products. For a more specific approach, the PCR products of 16S rDNA were digested with restriction enzymes. Seven of the samples, including type-strain ATCC 12472, were amplified by the hilA primers; these were subsequently sequenced. Gene yscD was amplified only in type-strain ATCC 12472. MspI and AluI digestion revealed 16S rDNA polymorphisms. This data allowed the generation of a dendogram for each analysis. The isolates of C. violaceum have variability in random genomic regions demonstrated by RAPD. Also, these isolates have variability in pathogenicity genes, as demonstrated by sequencing and restriction enzyme digestion.

Genetic characterization of strains of Saccharomycescerevisiae responsible for 'refermentation' in Botrytis-affected wines.

PubMed

Divol, B; Miot-Sertier, C; Lonvaud-Funel, A

2006-03-01

Saccharomyces cerevisiae is responsible for alcoholic fermentation of wines. However, some strains can also spoil sweet Botrytis-affected wines. Three 'refermentation' strains were isolated during maturation. Characterization of those strains in regards to their fingerprint, rDNA sequence and resistance to SO2, which constituted the main source of stress in Botrytis-affected wines, was carried out. Refermentation strains could be clearly discriminated by interdelta fingerprinting. However, they exhibited close relationships by karyotyping. A part of RDN1 locus sequence was examined by using PCR-RFLP and PCR-DGGE. The resistance of refermentation strains to SO2 was performed by using real time quantitative PCR focusing on SSU1 gene. Results suggested that refermentation strains were heterozygote in 26S rDNA and their ITS1-5.8S rDNA-ITS2 region sequence revealed relationships with 'flor' strains. As described in the literature for flor strain, two out of three refermentation strains constitutively developed a higher level of SSU1 expression than the reference strains, improving their putative tolerance to SO2. Therefore, refermentation strains of S. cerevisiae had developed many strategies to survive during maturing sweet wines. Singularities in rDNA sequence and SSU1 overexpression revealed a natural adaptation. Moreover, genomic relationship between flor and refermentation strains suggested that stress sources could induced selection of survivor strains.

Complete Genome Sequence of Methylobacterium populi P-1M, Isolated from Pink-Pigmented Household Biofilm.

PubMed

Morohoshi, Tomohiro; Ikeda, Tsukasa

2016-06-16

Methylobacterium populi P-1M is isolated from the pink-pigmented household biofilm. Here, we present the complete genome sequence of P-1M, consisting of one chromosome of 5,705,640 bp and five plasmids of 64,864 bp, 59,879 bp, 42,569 bp, 41,417 bp, and 29,506 bp. Copyright © 2016 Morohoshi and Ikeda.

Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran.

PubMed

Eslami, Ali; Meshgi, Behnam; Jalousian, Fatemeh; Rahmani, Shima; Salari, Mohammad Ali

2016-02-01

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.

Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: a genomic resource for studying agricultural pests.

PubMed

Noda, Hiroaki; Kawai, Sawako; Koizumi, Yoko; Matsui, Kageaki; Zhang, Qiang; Furukawa, Shigetoyo; Shimomura, Michihiko; Mita, Kazuei

2008-03-03

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is a serious insect pests of rice plants. Major means of BPH control are application of agricultural chemicals and cultivation of BPH resistant rice varieties. Nevertheless, BPH strains that are resistant to agricultural chemicals have developed, and BPH strains have appeared that are virulent against the resistant rice varieties. Expressed sequence tag (EST) analysis and related applications are useful to elucidate the mechanisms of resistance and virulence and to reveal physiological aspects of this non-model insect, with its poorly understood genetic background. More than 37,000 high-quality ESTs, excluding sequences of mitochondrial genome, microbial genomes, and rDNA, have been produced from 18 libraries of various BPH tissues and stages. About 10,200 clusters have been made from whole EST sequences, with average EST size of 627 bp. Among the top ten most abundantly expressed genes, three are unique and show no homology in BLAST searches. The actin gene was highly expressed in BPH, especially in the thorax. Tissue-specifically expressed genes were extracted based on the expression frequency among the libraries. An EST database is available at our web site. The EST library will provide useful information for transcriptional analyses, proteomic analyses, and gene functional analyses of BPH. Moreover, specific genes for hemimetabolous insects will be identified. The microarray fabricated based on the EST information will be useful for finding genes related to agricultural and biological problems related to this pest.

Zygosaccharomyces kombuchaensis, a new ascosporogenous yeast from 'Kombucha tea'.

PubMed

Kurtzman, C P; Robnett, C J; Basehoar-Powers, E

2001-07-01

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA. Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z. kombuchaensis near Zygosaccharomyces lentus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI.

Bacillus odysseyi isolate

NASA Technical Reports Server (NTRS)

La Duc, Myron Thomas (Inventor); Venkateswaran, Kasthuri (Inventor)

2007-01-01

The present invention relates to discovery and isolation of a biologically pure culture of a Bacillus odysseyi isolate with high adherence and sterilization resistant properties. B. odysseyi is a round spore forming Bacillus species that produces an exosporium. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and the type strain is 34hs-1.sup.T (=ATCC PTA-4993.sup.T=NRRL B-30641.sup.T=NBRC 100172.sup.T). The GenBank accession number for the 16S rDNA sequence of strain 34hs-1.sup.T is AF526913.

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

PubMed Central

Yang, Xue; Hameed, Uzma; Zhang, Ai-Fang; Zang, Hao-Yu; Gu, Chun-Yan; Chen, Yu; Xu, Yi-Liu

2017-01-01

Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity. PMID:28106107

Development of a PCR technique specific for Demodex injai in biological specimens.

PubMed

Sastre, N; Ravera, I; Ferreira, D; Altet, L; Sánchez, A; Bardagí, M; Francino, O; Ferrer, L

2013-09-01

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex-host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.

Identification of a Divergent Environmental DNA Sequence Clade Using the Phylogeny of Gregarine Parasites (Apicomplexa) from Crustacean Hosts

PubMed Central

Rueckert, Sonja; Simdyanov, Timur G.; Aleoshin, Vladimir V.; Leander, Brian S.

2011-01-01

Background Environmental SSU rDNA surveys have significantly improved our understanding of microeukaryotic diversity. Many of the sequences acquired using this approach are closely related to lineages previously characterized at both morphological and molecular levels, making interpretation of these data relatively straightforward. Some sequences, by contrast, appear to be phylogenetic orphans and are sometimes inferred to represent “novel lineages” of unknown cellular identity. Consequently, interpretation of environmental DNA surveys of cellular diversity rely on an adequately comprehensive database of DNA sequences derived from identified species. Several major taxa of microeukaryotes, however, are still very poorly represented in these databases, and this is especially true for diverse groups of single-celled parasites, such as gregarine apicomplexans. Methodology/Principal Findings This study attempts to address this paucity of DNA sequence data by characterizing four different gregarine species, isolated from the intestines of crustaceans, at both morphological and molecular levels: Thiriotia pugettiae sp. n. from the graceful kelp crab (Pugettia gracilis), Cephaloidophora cf. communis from two different species of barnacles (Balanus glandula and B. balanus), Heliospora cf. longissima from two different species of freshwater amphipods (Eulimnogammarus verrucosus and E. vittatus), and Heliospora caprellae comb. n. from a skeleton shrimp (Caprella alaskana). SSU rDNA sequences were acquired from isolates of these gregarine species and added to a global apicomplexan alignment containing all major groups of gregarines characterized so far. Molecular phylogenetic analyses of these data demonstrated that all of the gregarines collected from crustacean hosts formed a very strongly supported clade with 48 previously unidentified environmental DNA sequences. Conclusions/Significance This expanded molecular phylogenetic context enabled us to establish a major clade of intestinal gregarine parasites and infer the cellular identities of several previously unidentified environmental SSU rDNA sequences, including several sequences that have formerly been discussed broadly in the literature as a suspected “novel” lineage of eukaryotes. PMID:21483868

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species.

PubMed

Prado, E A; Faivre-Rampant, P; Schneider, C; Darmency, M A

1996-10-01

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii.

PubMed

Hamilton, P T; Reeve, J N

1985-01-01

DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29 bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5'GAANTTTCA and 5'TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heat-shock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5'AGGTGA.

Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

PubMed

Cooper, D N; Errington, L H; Clayton, R M

1983-01-01

Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches

PubMed Central

Kawano, Tetsuro; Imada, Mihoko; Chamavit, Pennapa; Kobayashi, Seiki; Hashimoto, Tetsuo

2017-01-01

Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba. PMID:28934335

Assessing the impact of fungicide enostroburin application on bacterial community in wheat phyllosphere.

PubMed

Gu, Likun; Bai, Zhihui; Jin, Bo; Hu, Qing; Wang, Huili; Zhuang, Guoqiang; Zhang, Hongxun

2010-01-01

Fungicides have been used extensively for controlling fungal pathogens of plants. However, little is known regarding the effects that fungicides upon the indigenous bacterial communities within the plant phyllosphere. The aims of this study were to assess the impact of fungicide enostroburin upon bacterial communities in wheat phyllosphere. Culture-independent methodologies of 16S rDNA clone library and 16S rDNA directed polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) were used for monitoring the change of bacterial community. The 16S rDNA clone library and PCR-DGGE analysis both confirmed the microbial community of wheat plant phyllosphere were predominantly of the gamma-Proteobacteria phyla. Results from PCR-DGGE analysis indicated a significant change in bacterial community structure within the phyllosphere following fungicide enostroburin application. Bands sequenced within control cultures were predominantly of Pseudomonas genus, but those bands sequenced in the treated samples were predominantly strains of Pantoea genus and Pseudomonas genus. Of interest was the appearance of two DGGE bands following fungicide treatment, one of which had sequence similarities (98%) to Pantoea sp. which might be a competitor of plant pathogens. This study revealed the wheat phyllosphere bacterial community composition and a shift in the bacterial community following fungicide enostroburin application.

Molecular evidence for a uniform microbial community in sponges from different oceans.

PubMed

Hentschel, Ute; Hopke, Jörn; Horn, Matthias; Friedrich, Anja B; Wagner, Michael; Hacker, Jörg; Moore, Bradley S

2002-09-01

Sponges (class Porifera) are evolutionarily ancient metazoans that populate the tropical oceans in great abundances but also occur in temperate regions and even in freshwater. Sponges contain large numbers of bacteria that are embedded within the animal matrix. The phylogeny of these bacteria and the evolutionary age of the interaction are virtually unknown. In order to provide insights into the species richness of the microbial community of sponges, we performed a comprehensive diversity survey based on 190 sponge-derived 16S ribosomal DNA (rDNA) sequences. The sponges Aplysina aerophoba and Theonella swinhoei were chosen for construction of the bacterial 16S rDNA library because they are taxonomically distantly related and they populate nonoverlapping geographic regions. In both sponges, a uniform microbial community was discovered whose phylogenetic signature is distinctly different from that of marine plankton or marine sediments. Altogether 14 monophyletic, sponge-specific sequence clusters were identified that belong to at least seven different bacterial divisions. By definition, the sequences of each cluster are more closely related to each other than to a sequence from nonsponge sources. These monophyletic clusters comprise 70% of all publicly available sponge-derived 16S rDNA sequences, reflecting the generality of the observed phenomenon. This shared microbial fraction represents the smallest common denominator of the sponges investigated in this study. Bacteria that are exclusively found in certain host species or that occur only transiently would have been missed. A picture emerges where sponges can be viewed as highly concentrated reservoirs of so far uncultured and elusive marine microorganisms.

ISSLS PRIZE IN CLINICAL SCIENCE 2017: Is infection the possible initiator of disc disease? An insight from proteomic analysis.

PubMed

Rajasekaran, S; Tangavel, Chitraa; Aiyer, Siddharth N; Nayagam, Sharon Miracle; Raveendran, M; Demonte, Naveen Luke; Subbaiah, Pramela; Kanna, Rishi; Shetty, Ajoy Prasad; Dharmalingam, K

2017-05-01

Proteomic and 16S rDNA analysis of disc tissues obtained in vivo. To address the controversy of infection as an aetiology for disc disorders through protein profiling. There is raging controversy over the presence of bacteria in human lumbar discs in vivo, and if they represent contamination or infection. Proteomics can provide valuable insight by identifying proteins signifying bacterial presence and, also host defence response proteins (HDRPs), which will confirm infection. 22 discs (15-disc herniations (DH), 5-degenerate (DD), 2-normal in MRI (NM) were harvested intraoperatively and immediately snap frozen. Samples were pooled into three groups and proteins extracted were analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Post identification, data analysis was performed using Uniprotdb, Pantherdb, Proteome discoverer and STRING network. Authentication for bacterial presence was performed by PCR amplification of 16S rDNA. LC-MS/MS analysis using Orbitrap showed 1103 proteins in DH group, compared to 394 in NM and 564 in DD. 73 bacterial specific proteins were identified (56 specific for Propionibacterium acnes; 17 for Staphylococcus epidermidis). In addition, 67 infection-specific HDRPs, unique or upregulated, such as Defensin, Lysozyme, Dermcidin, Cathepsin-G, Prolactin-Induced Protein, and Phospholipase-A2, were identified confirming presence of infection. Species-specific primers for P. acnes exhibited amplicons at 946 bp (16S rDNA) and 515 bp (Lipase) confirming presence of P. acnes in both NM discs, 11 of 15 DH discs, and all five DD discs. Bioinformatic search for protein-protein interactions (STRING) documented 169 proteins with close interactions (protein clustering co-efficient 0.7) between host response and degenerative proteins implying that infection may initiate degradation through Ubiquitin C. Our study demonstrates bacterial specific proteins and host defence proteins to infection which strengthen the hypothesis of infection as a possible initiator of disc disease. These results can lead to a paradigm shift in our understanding and management of disc disorders.

Complete mitochondrial genome of the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae).

PubMed

Kim, Min Jee; Im, Hyun Hwak; Lee, Kwang Youll; Han, Yeon Soo; Kim, Iksoo

2014-06-01

Abstract The complete nucleotide sequences of the mitochondrial genome from the whiter-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Scarabaeidae), was determined. The 20,319-bp long circular genome is the longest among completely sequenced Coleoptera. As is typical in animals, the P. brevitarsis genome consisted of two ribosomal RNAs, 22 transfer RNAs, 13 protein-coding genes and one A + T-rich region. Although the size of the coding genes was typical, the non-coding A + T-rich region was 5654 bp, which is the longest in insects. The extraordinary length of this region was composed of 28,117-bp tandem repeats and 782-bp tandem repeats. These repeat sequences were encompassed by three non-repeat sequences constituting 1804 bp.

First report of the epiphytic benthic dinoflagellates Coolia canariensis and Coolia malayensis in the waters off Jeju Island, Korea: morphology and rDNA sequences.

PubMed

Jeong, Hae Jin; Yih, Wonho; Kang, Nam Seon; Lee, Sung Yeon; Yoon, Eun Young; Yoo, Yeong Du; Kim, Hyung Seop; Kim, Jong Hyeok

2012-01-01

Coolia spp. are epiphytic and benthic dinoflagellates. Herein, we report for the first time, the occurrence of Coolia canariensis and Coolia malayensis in Korean waters. The morphology of the Korean strains of C. canariensis and C. malayensis isolated from the waters off Jeju Island, Korea was similar to that of the original Canary lslands strains and Malaysian strains, respectively. We found several pores and a line of small knobs on the pore plate, and perforations within the large pores of both C. canariensis and C. malayensis. The plates of the Korean strains of C. canariensis and C. malayensis were arranged in a Kofoidian series of Po, 3', 7'', 6c, 6s, 5''', and 2'''', and Po, 3', 7'', 7c, 6-7s, 5''', and 2'''', respectively. When properly aligned, the large subunit (LSU) rDNA sequence of the Korean strain of C. canariensis was identical to that of the Biscayan strains, but it was 2-3% different from the Canary lslands strain VGO0775 and the Australian strain. In addition, the sequences of small subunit (SSU) and/or LSU rDNA from the two Korean strains of C. malayensis were < 1% different from the Malaysian strains of C. malayensis and the Florida strain CCMP1345 and New Zealand strain CAWD39 ("Coolia monotis"). In phylogenetic trees based on LSU rDNA sequences, the Korean strains of C. malayensis belonged to a clade including the Malaysian strains and these two strains. Therefore, based on genealogical analyses, we suggest that the Korean strain of C. canariensis is closely related to two Atlantic strains and the Australian strain, whereas the Korean strains of C. malayensis are related to the Malaysian strains of C. malayensis and the Florida and New Zealand strains. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

PubMed Central

Garbaj, Aboubaker M.; Awad, Enas M.; Azwai, Salah M.; Abolghait, Said K.; Naas, Hesham T.; Moawad, Ashraf A.; Gammoudi, Fatim T.; Barbieri, Ilaria; Eldaghayes, Ibrahim M.

2016-01-01

Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow’s milk (11%), 3 isolates from she-camel’s milk (11%), two isolates from goat’s milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya. PMID:27956766

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

PubMed Central

2011-01-01

Background Ribosomal 5S genes are well known for the critical role they play in ribosome folding and functionality. These genes are thought to evolve in a concerted fashion, with high rates of homogenization of gene copies. However, the majority of previous analyses regarding the evolutionary process of rDNA repeats were conducted in invertebrates and plants. Studies have also been conducted on vertebrates, but these analyses were usually restricted to the 18S, 5.8S and 28S rRNA genes. The recent identification of divergent 5S rRNA gene paralogs in the genomes of elasmobranches and teleost fishes indicate that the eukaryotic 5S rRNA gene family has a more complex genomic organization than previously thought. The availability of new sequence data from lower vertebrates such as teleosts and elasmobranches enables an enhanced evolutionary characterization of 5S rDNA among vertebrates. Results We identified two variant classes of 5S rDNA sequences in the genomes of Potamotrygonidae stingrays, similar to the genomes of other vertebrates. One class of 5S rRNA genes was shared only by elasmobranches. A broad comparative survey among 100 vertebrate species suggests that the 5S rRNA gene variants in fishes originated from rounds of genome duplication. These variants were then maintained or eliminated by birth-and-death mechanisms, under intense purifying selection. Clustered multiple copies of 5S rDNA variants could have arisen due to unequal crossing over mechanisms. Simultaneously, the distinct genome clusters were independently homogenized, resulting in the maintenance of clusters of highly similar repeats through concerted evolution. Conclusions We believe that 5S rDNA molecular evolution in fish genomes is driven by a mixed mechanism that integrates birth-and-death and concerted evolution. PMID:21627815

FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

EPA Science Inventory

Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

Use of Fe(III) as an electron acceptor to recover previously uncultured hyperthermophiles: isolation and characterization of Geothermobacterium ferrireducens gen. nov., sp. nov.

PubMed

Kashefi, Kazem; Holmes, Dawn E; Reysenbach, Anna-Louise; Lovley, Derek R

2002-04-01

It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 microm by 1.0 to 1.2 microm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100 degrees C with an optimum of 85 to 90 degrees C. To our knowledge this is the highest temperature optimum of any organism in the Bacteria. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria. The name Geothermobacterium ferrireducens gen. nov., sp. nov., is proposed (ATCC BAA-426).

Application of rDNA-PCR amplification and DGGE fingerprinting for detection of microbial diversity in a Malaysian crude oil.

PubMed

Liew, Pauline Woanying; Jong, Bor Chyan

2008-05-01

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.

Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA.

PubMed

Sum, Jia-Siang; Lee, Wenn-Chyau; Amir, Amirah; Braima, Kamil A; Jeffery, John; Abdul-Aziz, Noraishah M; Fong, Mun-Yik; Lau, Yee-Ling

2014-07-03

Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene. Mosquitoes were collected, identified, dissected to check infection status, and DNA extraction was performed for PCR with primers targeting the ITS2 rDNA region. Sequencing was done and phylogenetic tree was constructed to study the evolutionary relationship among Anopheles mosquitoes within Peninsular Malaysia, as well as across the Asian region. A total of 133 Anopheles mosquitoes consisting of six different species were collected from eight different locations across Peninsular Malaysia. Of these, 65 ITS2 rDNA sequences were obtained. The ITS2 rDNA amplicons of the studied species were of different sizes. One collected species, Anopheles sinensis, shows two distinct pools of population in Peninsular Malaysia, suggesting evolvement of geographic race or allopatric speciation. Anopheles mosquitoes from Peninsular Malaysia show close evolutionary relationship with the Asian anophelines. Nevertheless, genetic differences due to geographical segregation can be seen. Meanwhile, some Anopheles mosquitoes in Peninsular Malaysia show vicariance, exemplified by the emergence of distinct cluster of An. sinensis population.

Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA

PubMed Central

2014-01-01

Background Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene. Methods Mosquitoes were collected, identified, dissected to check infection status, and DNA extraction was performed for PCR with primers targeting the ITS2 rDNA region. Sequencing was done and phylogenetic tree was constructed to study the evolutionary relationship among Anopheles mosquitoes within Peninsular Malaysia, as well as across the Asian region. Results A total of 133 Anopheles mosquitoes consisting of six different species were collected from eight different locations across Peninsular Malaysia. Of these, 65 ITS2 rDNA sequences were obtained. The ITS2 rDNA amplicons of the studied species were of different sizes. One collected species, Anopheles sinensis, shows two distinct pools of population in Peninsular Malaysia, suggesting evolvement of geographic race or allopatric speciation. Conclusion Anopheles mosquitoes from Peninsular Malaysia show close evolutionary relationship with the Asian anophelines. Nevertheless, genetic differences due to geographical segregation can be seen. Meanwhile, some Anopheles mosquitoes in Peninsular Malaysia show vicariance, exemplified by the emergence of distinct cluster of An. sinensis population. PMID:24993022

Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana

PubMed Central

Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe

2018-01-01

Abstract Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization. PMID:29518237

Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana.

PubMed

Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe; Probst, Aline V

2018-04-06

Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization.

Gymnotus coatesi (Gymnotiformes): A Case of Colocation of Multiple Sites of 18S rDNA with Telomeric Sequences.

PubMed

Machado, Milla de Andrade; Cardoso, Adauto Lima; Milhomem-Paixão, Susana Suely Rodrigues; Pieczarka, Julio Cesar; Nagamachi, Cleusa Yoshiko

2017-10-01

Gymnotus coatesi is a small and rare species of banded knife fish that was originally described by LaMonte in 1935, found along the main stretch of the Amazon River. There is no described cytogenetic data on this species. We analyzed the karyotype of five specimens of G. coatesi collected from Cururutuia Stream in Bragança, Pará, Brazil. The obtained diploid number is 50 and the karyotypic formula is 24 m/sm +26 st/a. The constitutive heterochromatin is DAPI positive and distributed mainly in the centromeric and pericentromeric regions of the chromosomes. Ag-nucleolus organizer regions staining showed nine active sites. The 5S rDNA probe hybridized chromosome pair 17 in the interstitial part of the long arm. Fluorescence in situ hybridization (FISH) with telomeric probes revealed signals only at terminal regions of the chromosomes. The 18S rDNA probe hybridized to 21 sites, and these signals colocalized with the telomeric sequences. This relatively high number of 18S rDNA sites may reflect gene duplication mediated by transposable elements. These results indicate that although the diploid number of G. coatesi is within the range previously observed for other members of the genus, various karyotypic characteristics distinguish G. coatesi from the other species of the genus and members of the Gymnotiform order.

Sinophysis and Pseudophalacroma are distantly related to typical Dinophysoid dinoflagellates (Dinophysales, Dinophyceae).

PubMed

Gómez, Fernando; Moreira, David; López-García, Purificación

2012-01-01

Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Dinophysoid dinoflagellates are usually considered a large monophyletic group. Large subunit and small subunit (SSU) rDNA phylogenies suggest a basal position for Amphisoleniaceae (Amphisolenia,Triposolenia) with respect to two sister groups, one containing most Phalacroma species plus Oxyphysis and the other Dinophysis,Ornithocercus, Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. Histioneis,Citharistes and some Phalacroma species. We provide here new SSU rDNA sequences of Pseudophalacroma (pelagic) and Sinophysis (the only benthic dinophysoid genus). Molecular phylogenies support that they are very divergent with respect to the main clade of Dinophysales. Additional molecular markers of these two key genera are needed to elucidate the evolutionary relations among the dinophysoid dinoflagellates. © 2011 The Author(s) Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

Hammondia sp. oocysts shed by a Brazilian fox (Lycalopex vetulus) differ from Hammondia heydorni and Hammondia triffittae.

PubMed

Gondim, Luís F P; Soares, Rodrigo M; Osaki, Silvia C; Snak, Alessandra; Grillo, Laura R; Fernandes, Nelson L M; de Carvalho, Anderson L

2018-05-21

A Brazilian fox (Lycalopex vetulus) was rescued from a highway, and 16 days after maintained in captivity, the fox shed oocysts with sizes compatible with Hammondia sp. and Neospora caninum. DNA extracted from oocysts were initially tested in two PCRs targeting the internal transcribed spacer 1 (ITS-1) of the rDNA of Hammondia heydorni and the Nc-5 gene of N. caninum. A 270-bp product was visualized in the PCR for H. heydorni. No amplification was observed for N. caninum PCR. Since ITS-1-based PCR is not sufficient to differentiate Hammondia species derived from canids, oocyst DNA was examined using multilocus sequence analysis of five genetic fragments [intron 1 of the alpha tubulin gene (intron 1), internal transcribed spaces 1 and 2 (ITS-1 and ITS-2) of the rDNA, 28S rRNA gene (D2/D3 domain), and heat shock protein 70 (Hsp70)]. The Hammondia sp. oocyst from the Brazilian fox, referred here as H-FOXBR isolate, is closely related to H. heydorni and Hammondia triffittae, but differs from these parasites in three genetic markers (alpha tubulin gene, ITS-2, and 28S rRNA). As reported by other research groups, Hammondia spp. excreted by canids are genetically diverse and may encompass additional species besides H. heydorni and H. triffittae. In this study, we confirmed that H-FOXBR has significant genetic differences in comparison to H. heydorni and H. triffittae and may represent a separate species. Further studies are needed to identify the life cycle of this parasite and to characterize the parasite stages in the intermediate and definitive hosts.

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide.

PubMed

Rachman, C N; Kabadjova, P; Prévost, H; Dousset, X

2003-01-01

The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.

Vertical distribution of the subsurface microorganisms in Sagara oil reservoir

NASA Astrophysics Data System (ADS)

Nunoura, T.; Oida, H.; Masui, N.; Ingaki, F.; Takai, K.; Nealson, K. H.; Horikoshi, K.

2002-12-01

The recent microbiological studies reported that active microbial habitat for methanogen, sulfate reducers (Archaeoglobus, d-Proteobacteria, gram positives), fermenters (Thermococcus, Thermotogales, gram positives etc.) and other heterotrophs (g-Proteobacteria etc.) are in subsurface petroleum oil reservoirs. However, microbial distribution at vertical distances in depth has not been demonstrated since the samples in previous studies are only to use oil and the formation water. Here, we show the vertical profile of microbial community structure in Japanese terrestrial oil reservoir by a combination of molecular ecological analyses and culture dependent studies. The sequential WRC (Whole Round Core) samples (200 mbsf) were recovered from a drilling project for Sagara oil reservoir, Shizuoka Prefecture, Japan, conducted in Jar. -Mar. 2002. The lithology of the core samples was composed of siltstone, sandstone, or partially oil containing sand. The major oil components were gasoline, kerosene and light oil, that is a unique feature observed in the Sagara oil reservoir. The direct count of DAPI-stained cells suggested that the biomass was relatively constant, 1.0x104cells/g through the core of the non-oil layers, whereas the oil-bearing layers had quite higher population density at a range of 1.0x105 ? 3.7x107cells/g. The vertical profile of microbial community structures was analyzed by the sequence similarity analysis, phylogenetic analysis and T-RFLP fingerprinting of PCR-amplified 16S rDNA. From bacterial rDNA clone libraries, most of the examined rDNA were similar with the sequence of genera Pseudomanas, Stenotrophomonas and Sphingomonas within g-Proteobacteria. Especially, Pseudomonas stutzeri was predominantly present in all oil-bearing layers. From archaeal rDNA clone libraries, all rDNA clone sequences were phylogenetically associated with uncultured soil group in Crenarchaeota. We detected none of the sequences of sulfate reducers, sulfur dependent fermenters and methanogens that have been previously detected as dominant microbial components in other oil reservoir environments. The absence of methanogen was consistent with the results from the stable isotopic analysis that major hydrocarbon components including methane in Sagara oil reservoir are thermogenic origin. In this presentation, we will also show the activity of the subsurface microbial components by the cultivation assays and discuss about the relationship between the microbial community structure and the formation process of petroleum in Sagara oil reservoir.

PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats.

PubMed

Guillen, I A; Camacho, H; Tuero, A D; Bacardí, D; Palenzuela, D O; Aguilera, A; Silva, J A; Estrada, R; Gell, O; Suárez, J; Ancizar, J; Brown, E; Colarte, A B; Castro, J; Novoa, L I

2016-09-01

The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies.

High Bacterial Diversity in Permanently Cold Marine Sediments

PubMed Central

Ravenschlag, Katrin; Sahm, Kerstin; Pernthaler, Jakob; Amann, Rudolf

1999-01-01

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the γ subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones. PMID:10473405

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

NASA Astrophysics Data System (ADS)

Su, Lei; Zhang, Qianqian; Gong, Jun

2017-07-01

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

Nuclear and mitochondrial rDNA variability in Crinipellis perniciosa from different geographic origins and hosts.

PubMed

de Arruda, Maricília C C; Ferreira, Marisa A S V; Miller, Robert N G; Resende, Mário Lúcio V; Felipe, Maria Sueli S

2003-01-01

Genetic variability in Crinipellis perniciosa, the causal organism of witches' broom disease in Theobroma cacao, was determined in strains originating from T. cacao and other susceptible host species Heteropterys acutifolia and Solanum lycocarpum in Brazil, in order to clarify host specificity and geographical variability. RFLP analysis of the ribosomal DNA ITS regions (rDNA ITS), and the mitochondrial DNA small subunit ribosomal DNA gene (mtDNA SSU rDNA) did not reveal any genetic variability in 120 tested strains, possibly serving only as species level markers. Genetic variability was observed in the ribosomal DNA IGS spacer region, in terms of IGS size, RFLPs and sequence data. Phylogenetic analyses (using CLUSTAL W, PHYLIP and TREEVIEW) indicated considerable differences between C. perniciosa strains from T. cacao and those from H. acutifolia (85-86%) and S. lycocarpum (95-96%). Sequence differences also indicated that C. perniciosa from T. cacao in Bahia is less variable (98%) when compared to the pathogen on T. cacao in Amazonas (97-98%), perhaps reflecting a recent introduction to T. cacao in Bahia.

Molecular application for identification of polycyclic aromatic hydrocarbons degrading bacteria (PAHD) species isolated from oil polluted soil in Dammam, Saud Arabia.

PubMed

Ibrahim, Mohamed M; Al-Turki, Ameena; Al-Sewedi, Dona; Arif, Ibrahim A; El-Gaaly, Gehan A

2015-09-01

Soil contamination with petroleum hydrocarbon products such as diesel and engine oil is becoming one of the major environmental problems. This study describes hydrocarbons degrading bacteria (PHAD) isolated from long-standing petrol polluted soil from the eastern region, Dammam, Saudi Arabia. The isolated strains were firstly categorized by accessible shape detection, physiological and biochemistry tests. Thereafter, a technique established on the sequence analysis of a 16S rDNA gene was used. Isolation of DNA from the bacterial strains was performed, on which the PCR reaction was carried out. Strains were identified based on 16S rDNA sequence analysis, As follows amplified samples were spontaneously sequenced automatically and the attained results were matched to open databases. Among the isolated bacterial strains, S1 was identified as Staphylococcus aureus and strain S1 as Corynebacterium amycolatum.

Karyotype Analysis of Four Vicia Species using In Situ Hybridization with Repetitive Sequences

PubMed Central

NAVRÁTILOVÁ, ALICE; NEUMANN, PAVEL; MACAS, JIŘÍ

2003-01-01

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S–25S and 5S rDNA, telomeres) and genus‐specific satellite repeats (VicTR‐A and VicTR‐B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR‐A and ‐B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied. PMID:12770847

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

PubMed Central

McGowan, Catherine; Fulthorpe, Roberta; Wright, Alice; Tiedje, J. M.

1998-01-01

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient. PMID:9758850

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae)

PubMed Central

Rosato, Marcela; Álvarez, Inés; Nieto Feliner, Gonzalo

2017-01-01

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation. PMID:29088249

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

PubMed

Rosato, Marcela; Álvarez, Inés; Nieto Feliner, Gonzalo; Rosselló, Josep A

2017-01-01

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Do neighboring lakes share common taxa of bacterioplankton? Comparison of 16S rDNA fingerprints and sequences from three geographic regions.

PubMed

Lindström, E S; Leskinen, E

2002-07-01

Bacterioplankton community composition was studied in 12 lakes in three different geographic regions in Scandinavia using denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rDNA. Area-specific abundant taxa were found in the lakes in two of the regions. In the region of Uppland the lakes had an alpha-proteobacterium, belonging to the subgroup Alpha V in common. The Alpha V bacteria appeared to be favored by neutral or higher pH values. The lakes in Lappland were found to harbor Actinobacteria, which appeared to be favored in bog lakes. No abundant taxon was found to be in common for the lakes in Svalbard, the third region studied.

Impact of cultivation on characterisation of species composition of soil bacterial communities.

PubMed

McCaig, A E.; Grayston, S J.; Prosser, J I.; Glover, L A.

2001-03-01

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Research on wind field algorithm of wind lidar based on BP neural network and grey prediction

NASA Astrophysics Data System (ADS)

Chen, Yong; Chen, Chun-Li; Luo, Xiong; Zhang, Yan; Yang, Ze-hou; Zhou, Jie; Shi, Xiao-ding; Wang, Lei

2018-01-01

This paper uses the BP neural network and grey algorithm to forecast and study radar wind field. In order to reduce the residual error in the wind field prediction which uses BP neural network and grey algorithm, calculating the minimum value of residual error function, adopting the residuals of the gray algorithm trained by BP neural network, using the trained network model to forecast the residual sequence, using the predicted residual error sequence to modify the forecast sequence of the grey algorithm. The test data show that using the grey algorithm modified by BP neural network can effectively reduce the residual value and improve the prediction precision.

[Microbial community structure in bio-ceramics and biological activated carbon analyzed by PCR-SSCP technique].

PubMed

Liu, Xiao-Lin; Liu, Wen-Jun

2007-04-01

Analyses of microbial community structure in bio-ceramics (BC) and biological activated carbon (BAC), which widely used in drinking water treatment were performed by polymerase-chain-reaction-single-strand-conformation-polymorphism (PCR-SSCP) targeted eubacterial 16S ribosomal RNA gene. Microorganisms on bio-ceramics and biological activated carbon were detached by ultrasonic, culturing on R2A and LB agar, respectively, followed by genome DNA extracting. Results show that larger than 10 kb genome DNA could be extracted from all the samples except the BAC samples processed by ultrasonic. However, quantities of the extracted DNA were different. 408 bp gene fragments were observed after PCR using the extracted genome DNA as templates. These gene fragments were digested with lambda exonuclease followed by SSCP electrophoresis. Same SSCP profiles were observed between ultrasonic eluting, R2A and LB agar culturing. The identity of the segment from bio-ceramics with uncultured Pseudomonas sp. Clone FTL201 16S rDNA (GenBank, AF509293.1) fragment was 92%, and identities of the two segments from BAC with Bacillus sp. JH19 16S rDNA (GenBank , DQ232748.1) fragment and Bacterium VA-S-11 16S rDNA (GenBank, AY395279.1) fragment were 100% and 99%, respectively.

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

PubMed Central

Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

2006-01-01

As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements. PMID:17069639

Complete mitochondrial genome of the larch hawk moth, Sphinx morio (Lepidoptera: Sphingidae).

PubMed

Kim, Min Jee; Choi, Sei-Woong; Kim, Iksoo

2013-12-01

The larch hawk moth, Sphinx morio, belongs to the lepidopteran family Sphingidae that has long been studied as a family of model insects in a diverse field. In this study, we describe the complete mitochondrial genome (mitogenome) sequences of the species in terms of general genomic features and characteristic short repetitive sequences found in the A + T-rich region. The 15,299-bp-long genome consisted of a typical set of genes (13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes) and one major non-coding A + T-rich region, with the typical arrangement found in Lepidoptera. The 316-bp-long A + T-rich region located between srRNA and tRNA(Met) harbored the conserved sequence blocks that are typically found in lepidopteran insects. Additionally, the A + T-rich region of S. morio contained three characteristic repeat sequences that are rarely found in Lepidoptera: two identical 12-bp repeat, three identical 5-bp-long tandem repeat, and six nearly identical 5-6 bp long repeat sequences.

The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

PubMed

Hamilton, John P; Neeno-Eckwall, Eric C; Adhikari, Bishwo N; Perna, Nicole T; Tisserat, Ned; Leach, Jan E; Lévesque, C André; Buell, C Robin

2011-01-01

The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera. Database URL: http://cpgr.plantbiology.msu.edu.

On the value of nuclear and mitochondrial gene sequences for reconstructing the phylogeny of vanilloid orchids (Vanilloideae, Orchidaceae)

PubMed Central

Cameron, Kenneth M.

2009-01-01

Background and Aims Most molecular phylogenetic studies of Orchidaceae have relied heavily on DNA sequences from the plastid genome. Nuclear and mitochondrial loci have only been superficially examined for their systematic value. Since 40% of the genera within Vanilloideae are achlorophyllous mycoheterotrophs, this is an ideal group of orchids in which to evaluate non-plastid gene sequences. Methods Phylogenetic reconstructions for Vanilloideae were produced using independent and combined data from the nuclear 18S, 5·8S and 26S rDNA genes and the mitochondrial atpA gene and nad1b-c intron. Key Results These new data indicate placements for genera such as Lecanorchis and Galeola, for which plastid gene sequences have been mostly unavailable. Nuclear and mitochondrial parsimony jackknife trees are congruent with each other and previously published trees based solely on plastid data. Because of high rates of sequence divergence among vanilloid orchids, even the short 5·8S rDNA gene provides impressive levels of resolution and support. Conclusions Orchid systematists are encouraged to sequence nuclear and mitochondrial gene regions along with the growing number of plastid loci available. PMID:19251715

Evaluation and validation of de novo and hybrid assembly techniques to derive high quality genome sequences

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn Marie; Land, Miriam L.; ...

2014-06-14

Our motivation with this work was to assess the potential of different types of sequence data combined with de novo and hybrid assembly approaches to improve existing draft genome sequences. Our results show Illumina, 454 and PacBio sequencing technologies were used to generate de novo and hybrid genome assemblies for four different bacteria, which were assessed for quality using summary statistics (e.g. number of contigs, N50) and in silico evaluation tools. Differences in predictions of multiple copies of rDNA operons for each respective bacterium were evaluated by PCR and Sanger sequencing, and then the validated results were applied as anmore » additional criterion to rank assemblies. In general, assemblies using longer PacBio reads were better able to resolve repetitive regions. In this study, the combination of Illumina and PacBio sequence data assembled through the ALLPATHS-LG algorithm gave the best summary statistics and most accurate rDNA operon number predictions. This study will aid others looking to improve existing draft genome assemblies. As to availability and implementation–all assembly tools except CLC Genomics Workbench are freely available under GNU General Public License.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE PAGES

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

2015-03-22

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

PubMed

Henry, Kelli F; Kawashima, Tomokazu; Goldberg, Robert B

2015-06-01

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

USDA-ARS?s Scientific Manuscript database

The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

Preliminary characterization of the oral microbiota of Chinese adults with and without gingivitis

PubMed Central

2011-01-01

Background Microbial communities inhabiting human mouth are associated with oral health and disease. Previous studies have indicated the general prevalence of adult gingivitis in China to be high. The aim of this study was to characterize in depth the oral microbiota of Chinese adults with or without gingivitis, by defining the microbial phylogenetic diversity and community-structure using highly paralleled pyrosequencing. Methods Six non-smoking Chinese, three with and three without gingivitis (age range 21-39 years, 4 females and 2 males) were enrolled in the present cross-sectional study. Gingival parameters of inflammation and bleeding on probing were characterized by a clinician using the Mazza Gingival Index (MGI). Plaque (sampled separately from four different oral sites) and salivary samples were obtained from each subject. Sequences and relative abundance of the bacterial 16 S rDNA PCR-amplicons were determined via pyrosequencing that produced 400 bp-long reads. The sequence data were analyzed via a computational pipeline customized for human oral microbiome analyses. Furthermore, the relative abundances of selected microbial groups were validated using quantitative PCR. Results The oral microbiomes from gingivitis and healthy subjects could be distinguished based on the distinct community structures of plaque microbiomes, but not the salivary microbiomes. Contributions of community members to community structure divergence were statistically accessed at the phylum, genus and species-like levels. Eight predominant taxa were found associated with gingivitis: TM7, Leptotrichia, Selenomonas, Streptococcus, Veillonella, Prevotella, Lautropia, and Haemophilus. Furthermore, 98 species-level OTUs were identified to be gingivitis-associated, which provided microbial features of gingivitis at a species resolution. Finally, for the two selected genera Streptococcus and Fusobacterium, Real-Time PCR based quantification of relative bacterial abundance validated the pyrosequencing-based results. Conclusions This methods study suggests that oral samples from this patient population of gingivitis can be characterized via plaque microbiome by pyrosequencing the 16 S rDNA genes. Further studies that characterize serial samples from subjects (longitudinal study design) with a larger population size may provide insight into the temporal and ecological features of oral microbial communities in clinically-defined states of gingivitis. PMID:22152152

In vitro transcription of a cloned mouse ribosomal RNA gene.

PubMed Central

Mishima, Y; Yamamoto, O; Kominami, R; Muramatsu, M

1981-01-01

An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system. Images PMID:6278446

Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

NASA Astrophysics Data System (ADS)

Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

2014-05-01

Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression

PubMed Central

Lisowski, Leszek; Lau, Ashley; Wang, Zhongya; Zhang, Yue; Zhang, Feijie; Grompe, Markus; Kay, Mark A

2012-01-01

Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8–13 times more frequently than control vectors, providing an estimate that 23–39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells. PMID:22990671

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers

PubMed Central

Udugama, Maheshi; Sanij, Elaine; Voon, Hsiao P. J.; Son, Jinbae; Hii, Linda; Henson, Jeremy D.; Chan, F. Lyn; Chang, Fiona T. M.; Liu, Yumei; Pearson, Richard B.; Kalitsis, Paul; Mann, Jeffrey R.; Collas, Philippe; Hannan, Ross D.; Wong, Lee H.

2018-01-01

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers. PMID:29669917

Molecular genetic identification of crustose representatives of the order Corallinales (Rhodophyta) in Chile.

PubMed

Vidal, Rodrigo; Meneses, Isabel; Smith, Macarena

2003-09-01

Knowledge on species of the order Corallinales along the coast of Chile is still scarce despite a number of studies and records of other divisions of seaweeds made since the early 20th century. This lack of information is more dramatic among crustose representatives of the order, thus depriving biogeographic studies of a thorough analysis and resulting in inadequately representative accounts of biodiversity. The currently changing taxonomy of the group makes it difficult to identify and differentiate among taxa based on morphological and developmental characters. Therefore, the use of molecular tools has been adopted in this study in order to facilitate identification and comparison of crustose corallines collected at the rocky intertidal between 27 degrees and 48 degrees S along the Pacific temperate coast of South America. A sequence 600bp (in length) from the SSU-rDNA gene was used to identify five taxa to the genus level: Lithophyllum, Spongites, Mesophyllum, Synarthrophyton, and Leptophytum. In all cases, the genus distinction based on morphological characters coincide with designations based on variation in the ribosomal DNA gene sequence. Spongites is the most frequently occurring genus and is found in all localities sampled while the others appear occasionally. Taxa recognition at species level must be examined with caution considering that morphological variability is not well understood in Chile because the SSU-rDNA region sequence does not always stand alone as an unambiguous means of identifying all coralline species. In such cases, more rapidly evolving markers are needed. For example, sequences from the ITS (rDNA) region often provide greater resolution among closely related species and genera. However, the methodology presented here remains a useful tool for species-level identification.

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences.

PubMed

Amor, Nabil; Farjallah, Sarra; Salem, Mohamed; Lamine, Dia Mamadou; Merella, Paolo; Said, Khaled; Ben Slimane, Badreddine

2011-10-01

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n=60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene. Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries. Copyright © 2011 Elsevier Inc. All rights reserved.

Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

PubMed Central

Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

2001-01-01

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

[Molecular identification of astragali radix and its adulterants by ITS sequences].

PubMed

Cui, Zhan-Hu; Li, Yue; Yuan, Qing-Jun; Zhou, Li-She; Li, Min-Hui

2012-12-01

To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence. Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4. ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants. ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease.

PubMed

Zuriaga, María Angeles; Mas-Coma, Santiago; Bargues, María Dolores

2015-05-01

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

The crustacean parasites Ellobiopsis Caullery, 1910 and Thalassomyces Niezabitowski, 1913 form a monophyletic divergent clade within the Alveolata.

PubMed

Gómez, Fernando; López-García, Purificación; Nowaczyk, Antoine; Moreira, David

2009-09-01

The Ellobiopsidae are enigmatic parasites of crustaceans that have been grouped together exclusively on the basis of morphological similarities. Ultrastructural studies have revealed their affiliation within the alveolates, which was confirmed by the phylogenetic analysis of the ribosomal RNA gene (SSU rDNA) sequences of two species of Thalassomyces Niezabitowski, 1913. However, their precise systematic position within this group remains unresolved, since they could not be definitively allied with any particular alveolate group. To better determine the systematic position of ellobiopsids by molecular phylogeny, we sequenced the SSU rDNA from the type-species of the Ellobiopsidae, Ellobiopsis chattoni Caullery, 1910. We found E. chattoni infecting various copepod hosts, Acartia clausi Giesbrecht, Centropages typicus Kröyer and Clausocalanus sp., in the Bay of Marseille, NW Mediterranean Sea, which allowed us to study several stages of the parasite development. A single unicellular multinucleate specimen provided two different sequences of the SSU rDNA gene, indicating the existence of polymorphism at this locus within single individuals. Ellobiopsis Caullery, 1910 and Thalassomyces formed a very divergent and well-supported clade in phylogenetic analyses. This clade appears to be more closely related to the dinoflagellates (including the Syndiniales/Marine Alveolate Group II and the Dinokaryota) and Marine Alveolate Group I than to the other alveolates (Ciliophora, Perkinsozoa and Apicomplexa).

The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis.

PubMed

Duan, Naibin; Sun, Honghe; Wang, Nan; Fei, Zhangjun; Chen, Xuesen

2016-07-01

The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis, a widely used apple rootstock, was determined using the Illumina high-throughput sequencing approach. The genome is 422,555 bp in length and has a GC content of 45.21%. It is separated by a pair of inverted repeats of 32,504 bp, to form a large single copy region of 213,055 bp and a small single copy region of 144,492 bp. The genome contains 38 protein-coding genes, four pseudogenes, 25 tRNA genes, and three rRNA genes. The genome is 25,608 bp longer than that of M. domestica, and several structural variations between these two mitogenomes were detected.

Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription

PubMed Central

Tsai, Yi-Tzang; Lin, Chen-I; Chen, Hung-Kai; Lee, Kuo-Ming; Hsu, Chia-Yi; Yang, Shun-Jen

2008-01-01

The short arms of five human acrocentric chromosomes contain ribosomal gene (rDNA) clusters where numerous mini-nucleoli arise at the exit of mitosis. These small nucleoli tend to coalesce into one or a few large nucleoli during interphase by unknown mechanisms. Here, we demonstrate that the N- and C-terminal domains of a nucleolar protein, hNopp140, bound respectively to α-satellite arrays and rDNA clusters of acrocentric chromosomes for nucleolar formation. The central acidic-and-basic repeated domain of hNopp140, possessing a weak self-self interacting ability, was indispensable for hNopp140 to build up a nucleolar round-shaped structure. The N- or the C-terminally truncated hNopp140 caused nucleolar segregation and was able to alter locations of the rDNA transcription, as mediated by detaching the rDNA repeats from the acrocentric α-satellite arrays. Interestingly, an hNopp140 mutant, made by joining the N- and C-terminal domains but excluding the entire central repeated region, induced nucleolar disruption and global chromatin condensation. Furthermore, RNAi knockdown of hNopp140 resulted in dispersion of the rDNA and acrocentric α-satellite sequences away from nucleolus that was accompanied by rDNA transcriptional silence. Our findings indicate that hNopp140, a scaffold protein, is involved in the nucleolar assembly, fusion, and maintenance. PMID:18253863

RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes.

PubMed

Begnis, Martina; Apte, Manasi S; Masuda, Hirohisa; Jain, Devanshi; Wheeler, David Lee; Cooper, Julia Promisel

2018-04-01

The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATI rDNA " chromosomes), it is dispensable for HAATI rDNA maintenance. Surprisingly, Dicer (Dcr1) plays a separate, RNAi-independent role in preventing formation of the rare HAATI subtype in which a different repetitive element (the subtelomeric element) replaces telomeres. Using genetics and fusions between shelterin components and rDNA-binding proteins, we mapped the mechanism by which rDNA loci engage crucial end protection factors-despite the absence of telomere repeats-and secure end protection. Sequence analysis of HAATI rDNA genomes allowed us to propose RNA and DNA polymerase template-switching models for the mechanism of RNAi-triggered rDNA translocations. Collectively, our results reveal unforeseen roles for noncoding RNAs (ncRNAs) in assembling a telomere-free chromosome end protection device. © 2018 Begnis et al.; Published by Cold Spring Harbor Laboratory Press.

Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: A genomic resource for studying agricultural pests

PubMed Central

Noda, Hiroaki; Kawai, Sawako; Koizumi, Yoko; Matsui, Kageaki; Zhang, Qiang; Furukawa, Shigetoyo; Shimomura, Michihiko; Mita, Kazuei

2008-01-01

Background The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is a serious insect pests of rice plants. Major means of BPH control are application of agricultural chemicals and cultivation of BPH resistant rice varieties. Nevertheless, BPH strains that are resistant to agricultural chemicals have developed, and BPH strains have appeared that are virulent against the resistant rice varieties. Expressed sequence tag (EST) analysis and related applications are useful to elucidate the mechanisms of resistance and virulence and to reveal physiological aspects of this non-model insect, with its poorly understood genetic background. Results More than 37,000 high-quality ESTs, excluding sequences of mitochondrial genome, microbial genomes, and rDNA, have been produced from 18 libraries of various BPH tissues and stages. About 10,200 clusters have been made from whole EST sequences, with average EST size of 627 bp. Among the top ten most abundantly expressed genes, three are unique and show no homology in BLAST searches. The actin gene was highly expressed in BPH, especially in the thorax. Tissue-specifically expressed genes were extracted based on the expression frequency among the libraries. An EST database is available at our web site. Conclusion The EST library will provide useful information for transcriptional analyses, proteomic analyses, and gene functional analyses of BPH. Moreover, specific genes for hemimetabolous insects will be identified. The microarray fabricated based on the EST information will be useful for finding genes related to agricultural and biological problems related to this pest. PMID:18315884

The first complete organellar genomes of an Antarctic red alga, Pyropia endiviifolia: insights into its genome architecture and phylogenetic position within genus Pyropia (Bangiales, Rhodophyta)

NASA Astrophysics Data System (ADS)

Xu, Kuipeng; Tang, Xianghai; Bi, Guiqi; Cao, Min; Wang, Lu; Mao, Yunxiang

2017-08-01

Pyropia species grow in the intertidal zone and are cold-water adapted. To date, most of the information about the whole plastid and mitochondrial genomes (ptDNA and mtDNA) of this genus is limited to Northern Hemisphere species. Here, we report the sequencing of the ptDNA and mtDNA of the Antarctic red alga Pyropia endiviifolia using the Illumina platform. The plastid genome (195 784 bp, 33.28% GC content) contains 210 protein-coding genes, 37 tRNA genes and 6 rRNA genes. The mitochondrial genome (34 603 bp, 30.5% GC content) contains 26 protein-coding genes, 25 tRNA genes and 2 rRNA genes. Our results suggest that the organellar genomes of Py. endiviifolia have a compact organization. Although the collinearity of these genomes is conserved compared with other Pyropia species, the genome sizes show significant differences, mainly because of the different copy numbers of rDNA operons in the ptDNA and group II introns in the mtDNA. The other Pyropia species have 2u20133 distinct intronic ORFs in their cox 1 genes, but Py. endiviifolia has no introns in its cox 1 gene. This has led to a smaller mtDNA than in other Pyropia species. The phylogenetic relationships within Pyropia were examined using concatenated gene sets from most of the available organellar genomes with both the maximum likelihood and Bayesian methods. The analysis revealed a sister taxa affiliation between the Antarctic species Py. endiviifolia and the North American species Py. kanakaensis.

Screening, Isolation and Identification of Probiotic Producing Lactobacillus acidophilus Strains EMBS081 & EMBS082 by 16S rRNA Gene Sequencing.

PubMed

Chandok, Harshpreet; Shah, Pratik; Akare, Uday Raj; Hindala, Maliram; Bhadoriya, Sneha Singh; Ravi, G V; Sharma, Varsha; Bandaru, Srinivas; Rathore, Pragya; Nayarisseri, Anuraj

2015-09-01

16S rDNA sequencing which has gained wide popularity amongst microbiologists for the molecular characterization and identification of newly discovered isolates provides accurate identification of isolates down to the level of sub-species (strain). Its most important advantage over the traditional biochemical characterization methods is that it can provide an accurate identification of strains with atypical phenotypic characters as well. The following work is an application of 16S rRNA gene sequencing approach to identify a novel species of Probiotic Lactobacillus acidophilus. The sample was collected from pond water samples of rural and urban areas of Krishna district, Vijayawada, Andhra Pradesh, India. Subsequently, the sample was serially diluted and the aliquots were incubated for a suitable time period following which the suspected colony was subjected to 16S rDNA sequencing. The sequence aligned against other species was concluded to be a novel, Probiotic L. acidophilus bacteria, further which were named L. acidophilus strain EMBS081 & EMBS082. After the sequence characterization, the isolate was deposited in GenBank Database, maintained by the National Centre for Biotechnology Information NCBI. The sequence can also be retrieve from EMBL and DDBJ repositories with accession numbers JX255677 and KC150145.

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis.

PubMed

Wang, Jun; Chen, Wen Feng; Li, Qing X

2012-02-24

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4-99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9-99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification. Copyright © 2011 Elsevier B.V. All rights reserved.

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil.

PubMed

Teixeira, D C; Wulff, N A; Martins, E C; Kitajima, E W; Bassanezi, R; Ayres, A J; Eveillard, S; Saillard, C; Bové, J M

2008-09-01

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species.

PubMed

Larsen, Svend Arild; Mogensen, Line; Dietz, Rune; Baagøe, Hans Jørgen; Andersen, Mogens; Werge, Thomas; Rasmussen, Henrik Berg

2005-12-01

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.

Identification of Simple Sequence Repeats in Chloroplast Genomes of Magnoliids Through Bioinformatics Approach.

PubMed

Srivastava, Deepika; Shanker, Asheesh

2016-12-01

Basal angiosperms or Magnoliids is an important clade of commercially important plants which mainly include spices and edible fruits. In this study, 17 chloroplast genome sequences belonging to clade Magnoliids were screened for the identification of chloroplast simple sequence repeats (cpSSRs). Simple sequence repeats or microsatellites are short stretches of DNA up to 1-6 base pair in length. These repeats are ubiquitous and play important role in the development of molecular markers and to study the mapping of traits of economic, medical or ecological interest. A total of 479 SSRs were detected, showing average density of 1 SSR/6.91 kb. Depending on the repeat units, the length of SSRs ranged from 12 to 24 bp for mono-, 12 to 18 bp for di-, 12 to 26 bp for tri-, 12 to 24 bp for tetra-, 15 bp for penta- and 18 bp for hexanucleotide repeats. Mononucleotide repeats were the most frequent (207, 43.21 %) followed by tetranucleotide repeats (130, 27.13 %). Penta- and hexanucleotide repeats were least frequent or absent in these chloroplast genomes.

Testing deep reticulate evolution in Amaryllidaceae Tribe Hippeastreae (Asparagales) with ITS and chloroplast sequence data

USDA-ARS?s Scientific Manuscript database

The phylogeny of Amaryllidaceae tribe Hippeastreae was inferred using chloroplast (3’ycf1, ndhF, trnL-F) and nuclear (ITS rDNA) sequence data under maximum parsimony and maximum likelihood frameworks. Network analyses were applied to resolve conflicting signals among data sets and putative scenarios...

Chromobacterium sphagni sp. nov., an insecticidal bacterium isolated from Sphagnum bogs

USDA-ARS?s Scientific Manuscript database

Sixteen isolates of Gram-negative, motile, violet-pigmented bacteria were isolated from Sphagnum bogs in West Virginia and Maine, USA. 16S rDNA sequences and fatty acid analysis (FAME) revealed a high degree of relatedness among the isolates, and genomic sequencing of two isolates, IIBBL 14B-1 and I...

Specific PCR for Myxobolus arcticus SSU rDNA in juvenile sockeye salmon Oncorhynchus nerka from British Columbia, Canada.

PubMed

Mahony, Amelia; Fraser, Sarah; Groman, David B; Jones, Simon R M

2015-06-29

A PCR for the specific detection of the salmon brain parasite Myxobolus arcticus (Pugachev and Khokhlov, 1979) was developed using primers designed to amplify a 1363 base pair fragment of the small subunit rDNA. The assay did not amplify DNA from 5 other Myxobolus species or from 7 other myxozoan species belonging to 5 other genera. For juvenile sockeye salmon Oncorhynchus nerka (Walbaum) collected from Chilko Lake, British Columbia (BC), Canada, in 2011, the prevalence by PCR was 96%, in contrast to 71% by histological examination of brain tissue. In 2010, the histological prevalence was 52.5%. Sequence identity between M. arcticus from Chilko Lake and other sites in BC ranged from 99.7 to 99.8% and was 99.6% for a Japanese sequence. In contrast, an M. arcticus sequence from Norway shared 95.3% identity with the Chilko Lake sequence, suggesting misidentification of the parasite. Chilko Lake sockeye salmon were previously reported free of infection with M. arcticus, and more research is required to understand the processes involved in the local and global dispersion of this parasite.

Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea).

PubMed

Tarcz, Sebastian; Potekhin, Alexey; Rautian, Maria; Przyboś, Ewa

2012-05-01

This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens. Copyright © 2012 Elsevier Inc. All rights reserved.

Eukaryotic Plankton Species Diversity in the Western Channel of the Korea Strait using 18S rDNA Sequences and its Implications for Water Masses

NASA Astrophysics Data System (ADS)

Lee, Sang-Rae; Song, Eun Hye; Lee, Tongsup

2018-03-01

Organisms entering the East Sea (Sea of Japan) through the Korea Strait, together with water, salt, and energy, affect the East Sea ecosystem. In this study, we report on the biodiversity of eukaryotic plankton found in the Western Channel of the Korea Strait for the first time using small subunit ribosomal RNA gene (18S rDNA) sequences. We also discuss the characteristics of water masses and their physicochemical factors. Diverse taxonomic groups were recovered from 18S rDNA clone libraries, including putative novel, higher taxonomic entities affiliated with Cercozoa, Raphidophyceae, Picozoa, and novel marine Stramenopiles. We also found that there was cryptic genetic variation at both the intraspecific and interspecific levels among arthropods, diatoms, and green algae. Specific plankton assemblages were identified at different sampling depths and they may provide useful information that could be used to interpret the origin and the subsequent mixing history of the water masses that contribute to the Tsushima Warm Current waters. Furthermore, the biological information highlighted in this study may help improve our understanding about the complex water mass interactions that were highlighted in the Korea Strait.

Internal and external relationships of the Cnidaria: implications of primary and predicted secondary structure of the 5'-end of the 23S-like rDNA.

PubMed Central

Odorico, D M; Miller, D J

1997-01-01

Since both internal (class-level) and external relationships of the Cnidaria remain unclear on the basis of analyses of 18S and (partial) 16S rDNA sequence data, we examined the informativeness of the 5'-end of the 23S-like rDNA. Here we describe analyses of both primary and predicted secondary structure data for this region from the ctenophore Bolinopsis sp., the placozoan Trichoplax adhaerens, the sponge Hymeniacidon heliophila, and representatives of all four cnidarian classes. Primary sequence analyses clearly resolved the Cnidaria from other lower Metazoa, supported sister group relationships between the Scyphozoa and Cubozoa and between the Ctenophora and the Placozoa, and confirmed the basal status of the Anthozoa within the Cnidaria. Additionally, in the ctenophore, placozoan and sponge, non-canonical base pairing is required to maintain the secondary structure of the B12 region, whereas amongst the Cnidaria this is not the case. Although the phylogenetic significance of this molecular character is unclear, our analyses do not support the close relationship between Cnidaria and Placozoa suggested by previous studies. PMID:9061962

Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization.

PubMed

Matoba, Hideyuki; Mizutani, Takayuki; Nagano, Katsuya; Hoshi, Yoshikazu; Uchiyama, Hiroshi

2007-12-01

In this study, in addition to the karyotype analysis, the chromosomal distributions of 5 S and 18 S rDNAs, and the Arabidopsis-type (T3AG3) telomeric sequences were detected by means of fluorescence in situ hybridization (FISH) to promote the information of chromosomal organization and evolution in the cultivated lettuce and its wild relatives, L. sativa, L. serriola, L. saligna and L. virosa. The karyotype analysis revealed the dissimilarity between L. virosa and the remaining species. In all four Lactuca species studied, one 5 S rDNA and two 18 S rDNA loci were detected. The simultaneous FISH of 5 S and 18 S rDNAs revealed that both rDNA loci of L. sativa, L. serriola and L. saligna were identical, however, that of L. virosa was different from the other species. These analyses indicate the closer relationships between L. sativa/L. serriola and L. saligna rather than L. virosa. Arabidopsis-type telomeric sequences were detected at both ends of their chromatids of all chromosomes not in the other regions. This observation suggests the lack of telomere-mediated chromosomal rearrangements among the Lactuca chromosomes.

Definition of Cis-Acting Elements Regulating Expression of the Drosophila Melanogaster Ninae Opsin Gene by Oligonucleotide-Directed Mutagenesis

PubMed Central

Mismer, D.; Rubin, G. M.

1989-01-01

We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter. PMID:2521839

A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae)

PubMed Central

2014-01-01

Background Poecilimon and Isophya are the largest genera of the tribe Barbitistini and among the most systematically complicated and evolutionarily intriguing groups of Palearctic tettigoniids. We examined the genomic organization of 79 taxa with a stable chromosome number using classical (C–banding, silver and fluorochrome staining) and molecular (fluorescence in situ hybridization with 18S rDNA and (TTAGG) n telomeric probes) cytogenetic techniques. These tools were employed to establish genetic organization and differences or similarities between genera or species within the same genus and determine if cytogenetic markers can be used for identifying some taxonomic groups of species. Results Differences between the karyotypes of the studied genera include some general changes in the morphology of the X chromosome in Isophya (in contrast to Poecilimon). The number of major rDNA clusters per haploid genome divided Poecilimon into two main almost equal groups (with either one or two clusters), while two rDNA clusters predominated in Isophya. In both genera, rDNA loci were preferentially located in the paracentromeric region of the autosomes and rarely in the sex chromosomes. Our results demonstrate a coincidence between the location of rDNA loci and active NORs and GC-rich heterochromatin regions. The C/DAPI/CMA3 bands observed in most Poecilimon chromosomes suggest the presence of more families of repetitive DNA sequences as compared to the heterochromatin patterns in Isophya. Conclusions The results show both differences and similarities in genome organization among species of the same genus and between genera. Previous views on the systematics and phylogenetic grouping of certain lineages are discussed in light of the present cytogenetic results. In some cases, variation of chromosome markers was observed to correspond with variation in other evolutionary traits, which is related to the processes of ongoing speciation and hybridization in zones of secondary contact. It was concluded that the physical mapping of rDNA sequences and heterochromatin may be used as an additional marker for understanding interspecific relationships in these groups and their routes of speciation. PMID:24625118

Localization of the 5S and 45S rDNA Sites and cpDNA Sequence Analysis in Species of the Quadrifaria Group of Paspalum (Poaceae, Paniceae)

PubMed Central

VAIO, MAGDALENA; SPERANZA, PABLO; VALLS, JOSÉ FRANCISCO; GUERRA, MARCELO; MAZZELLA, CRISTINA

2005-01-01

• Background and Aims The Quadrifaria group of Paspalum (Poaceae, Paniceae) comprises species native to the subtropical and temperate regions of South America. The purpose of this research was to characterize the I genomes in five species of this group and to establish phylogenetic relationships among them. • Methods Prometaphase chromatin condensation patterns, the physical location of 5S and 45S rDNA sites by fluorescence in situ hybridization (FISH), and sequences of five chloroplast non-coding regions were analysed. • Key Results The condensation patterns observed were highly conserved among diploid and tetraploid accessions studied and not influenced by the dyes used or by the FISH procedure, allowing the identification of almost all the chromosome pairs that carried the rDNA signals. The FISH analysis of 5S rDNA sites showed the same localization and a correspondence between the number of sites and ploidy level. In contrast, the distribution of 45S rDNA sites was variable. Two general patterns were observed with respect to the location of the 45S rDNA. The species and cytotypes Paspalum haumanii 2x, P. intermedium 2x, P. quadrifarium 4x and P. exaltatum 4x showed proximal sites on chromosome 8 and two to four distal sites in other chromosomes, while P. quarinii 4x and P. quadrifarium 2x showed only distal sites located on a variable number of small chromosomes and on the long arm of chromosome 1. The single most-parsimonious tree found from the phylogenetic analysis showed the Quadrifaria species partitioned in two clades, one of them includes P. haumanii 2x and P. intermedium 2x together with P. quadrifarium 4x and P. exaltatum 4x, while the other contains P. quadrifarium 2x and P. quarinii 4x. • Conclusions The subdivision found with FISH is consistent with the clades recovered with cpDNA data and both analyses suggest that the Quadrifaria group, as presently defined, is not monophyletic and its species belong in at least two clades. PMID:15911540

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

PubMed

de la Bastide, Paul Y; Leung, Wai Lam; Hintz, William E

2015-01-01

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Multiplex PCR identification of Taenia spp. in rodents and carnivores.

PubMed

Al-Sabi, Mohammad N S; Kapel, Christian M O

2011-11-01

The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

Bacteria of an anaerobic 1,2-dichloropropane-dechlorinating mixed culture are phylogenetically related to those of other anaerobic dechlorinating consortia.

PubMed

Schlötelburg, C; von Wintzingerode, F; Hauck, R; Hegemann, W; Göbel, U B

2000-07-01

A 16S-rDNA-based molecular study was performed to determine the bacterial diversity of an anaerobic, 1,2-dichloropropane-dechlorinating bioreactor consortium derived from sediment of the River Saale, Germany. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified using conserved primers. A clone library was constructed and analysed by sequencing the 16S rDNA inserts of randomly chosen clones followed by dot blot hybridization with labelled polynucleotide probes. The phylogenetic analysis revealed significant sequence similarities of several as yet uncultured bacterial species in the bioreactor to those found in other reductively dechlorinating freshwater consortia. In contrast, no close relationship was obtained with as yet uncultured bacteria found in reductively dechlorinating consortia derived from marine habitats. One rDNA clone showed >97% sequence similarity to Dehalobacter species, known for reductive dechlorination of tri- and tetrachloroethene. These results suggest that reductive dechlorination in microbial freshwater habitats depends upon a specific bacterial community structure.

Second-generation sequencing of entire mitochondrial coding-regions (∼15.4 kb) holds promise for study of the phylogeny and taxonomy of human body lice and head lice.

PubMed

Xiong, H; Campelo, D; Pollack, R J; Raoult, D; Shao, R; Alem, M; Ali, J; Bilcha, K; Barker, S C

2014-08-01

The Illumina Hiseq platform was used to sequence the entire mitochondrial coding-regions of 20 body lice, Pediculus humanus Linnaeus, and head lice, P. capitis De Geer (Phthiraptera: Pediculidae), from eight towns and cities in five countries: Ethiopia, France, China, Australia and the U.S.A. These data (∼310 kb) were used to see how much more informative entire mitochondrial coding-region sequences were than partial mitochondrial coding-region sequences, and thus to guide the design of future studies of the phylogeny, origin, evolution and taxonomy of body lice and head lice. Phylogenies were compared from entire coding-region sequences (∼15.4 kb), entire cox1 (∼1.5 kb), partial cox1 (∼700 bp) and partial cytb (∼600 bp) sequences. On the one hand, phylogenies from entire mitochondrial coding-region sequences (∼15.4 kb) were much more informative than phylogenies from entire cox1 sequences (∼1.5 kb) and partial gene sequences (∼600 to ∼700 bp). For example, 19 branches had > 95% bootstrap support in our maximum likelihood tree from the entire mitochondrial coding-regions (∼15.4 kb) whereas the tree from 700 bp cox1 had only two branches with bootstrap support > 95%. Yet, by contrast, partial cytb (∼600 bp) and partial cox1 (∼486 bp) sequences were sufficient to genotype lice to Clade A, B or C. The sequences of the mitochondrial genomes of the P. humanus, P. capitis and P. schaeffi Fahrenholz studied are in NCBI GenBank under the accession numbers KC660761-800, KC685631-6330, KC241882-97, EU219988-95, HM241895-8 and JX080388-407. © 2014 The Royal Entomological Society.

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees.

PubMed

Adams, Gerard C; Surve-Iyer, Rupa S; Iezzoni, Amy F

2002-01-01

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

High-Resolution Whole-Genome Sequencing Reveals That Specific Chromatin Domains from Most Human Chromosomes Associate with Nucleoli

PubMed Central

van Koningsbruggen, Silvana; Gierliński, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J.; Ariyurek, Yavuz; den Dunnen, Johan T.

2010-01-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope. PMID:20826608

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

PubMed

van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

2010-11-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Potato-associated arbuscular mycorrhizal fungal communities in the Peruvian Andes.

PubMed

Senés-Guerrero, Carolina; Torres-Cortés, Gloria; Pfeiffer, Stefan; Rojas, Mercy; Schüßler, Arthur

2014-08-01

The world's fourth largest food crop, potato, originates in the Andes. Here, the community composition of arbuscular mycorrhizal fungi (AMF) associated with potato in Andean ecosystems is described for the first time. AMF were studied in potato roots and rhizosphere soil at four different altitudes from 2,658 to 4,075 m above mean sea level (mamsl) and in three plant growth stages (emergence, flowering, and senescence). AMF species were distinguished by sequencing an approx. 1,500 bp nuclear rDNA region. Twenty species of AMF were identified, of which 12 came from potato roots and 15 from rhizosphere soil. Seven species were found in both roots and soil. Interestingly, altitude affected species composition with the highest altitude exhibiting the greatest species diversity. The three most common colonizers of potato roots detected were Funneliformis mosseae, an unknown Claroideoglomus sp., and Rhizophagus irregularis. Notably, the potato-associated AMF diversity observed in this Andean region is much higher than that reported for potato in other ecosystems. Potato plants were colonized by diverse species from 8 of the 11 Glomeromycota families. Identification of the AMF species is important for their potential use in sustainable management practices to improve potato production in the Andean region.

A new cultivation independent approach to detect and monitor common Trichoderma species in soils.

PubMed

Hagn, Alexandra; Wallisch, Stefanie; Radl, Viviane; Charles Munch, Jean; Schloter, Michael

2007-04-01

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.

Experimental infection of mice with tightly coiled spiral bacteria ("Candidatus Helicobacter suis") originating from the pig stomach.

PubMed

Park, J-H; Hong, J J; Park, J H

2003-01-01

Mice (n=34) were inoculated orally with a gastric homogenate from a pig infected with tightly coiled spiral bacteria (TCSB). In mice killed in pairs at 16 intervals up to 108 weeks post-inoculation (pi), TCSB were invariably found, mainly in the mucosal surface, gastric pits, intercellular spaces, cytoplasm of surface epithelial cells, and lumina of gastric glands. Histopathologically, infiltration of lymphocytes and plasma cells was seen from 8 weeks pi onwards, gradually increasing as infection progressed. From 64 weeks pi onwards, the formation of large follicles was observed in the lamina propria and submucosa, together with severe necrosis of surface epithelial cells. Glandular epithelial cells in the fundic mucosa were markedly dysplastic and intruded through the basement membrane into the submucosal layer. Common antigenicity between TCSB and Helicobacter pylori was demonstrated by Western blotting, ELISA, and immunohistochemistry. The sequence of the 16S rDNA fragment of 374 bp showed 100% homology with the 16S rRNA gene of "Candidatus Helicobacter suis". Experimental infection of the gastric mucosa of mice with TCSB was closely associated with chronic gastritis and dysplastic lesions.

Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH

PubMed Central

Gan, Yimei; Liu, Fang; Chen, Dan; Wu, Qiong; Qin, Qin; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

2013-01-01

We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A1D1). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G . incanum (E4), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G . raimondii (D5) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution. PMID:23826377

Chromosomal Locations of 5S and 45S rDNA in Gossypium Genus and Its Phylogenetic Implications Revealed by FISH.

PubMed

Gan, Yimei; Liu, Fang; Chen, Dan; Wu, Qiong; Qin, Qin; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

2013-01-01

We investigated the locations of 5S and 45S rDNA in Gossypium diploid A, B, D, E, F, G genomes and tetraploid genome (AD) using multi-probe fluorescent in situ hybridization (FISH) for evolution analysis in Gossypium genus. The rDNA numbers and sizes, and synteny relationships between 5S and 45S were revealed using 5S and 45S as double-probe for all species, and the rDNA-bearing chromosomes were identified for A, D and AD genomes with one more probe that is single-chromosome-specific BAC clone from G. hirsutum (A1D1). Two to four 45S and one 5S loci were found in diploid-species except two 5S loci in G. incanum (E4), the same as that in tetraploid species. The 45S on the 7th and 9th chromosomes and the 5S on the 9th chromosomes seemed to be conserved in A, D and AD genomes. In the species of B, E, F and G genomes, the rDNA numbers, sizes, and synteny relationships were first reported in this paper. The rDNA pattern agrees with previously reported phylogenetic history with some disagreements. Combined with the whole-genome sequencing data from G. raimondii (D5) and the conserved cotton karyotype, it is suggested that the expansion, decrease and transposition of rDNA other than chromosome rearrangements might occur during the Gossypium evolution.

Draft Genome Sequences of Acinetobacter and Bacillus Strains Isolated from Spacecraft-Associated Surfaces

PubMed Central

Seuylemezian, Arman; Vaishampayan, Parag; Cooper, Kerry

2018-01-01

ABSTRACT We report here the draft genome sequences of four strains isolated from spacecraft-associated surfaces exhibiting increased resistance to stressors such as UV radiation and exposure to H2O2. The draft genomes of strains 1P01SCT, FO-92T, 50v1, and 2P01AA had sizes of 5,500,894 bp, 4,699,376 bp, 3,174,402 bp, and 4,328,804 bp, respectively. PMID:29439046

Dead Element Replicating: Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect (Insecta: Phasmida)

PubMed Central

Martoni, Francesco; Eickbush, Danna G.; Scavariello, Claudia; Luchetti, Andrea; Mantovani, Barbara

2015-01-01

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution. PMID:25799008

Identification and genetic characterization of Sarcocystis arctica and Sarcocystis lutrae in red foxes (Vulpes vulpes) from Baltic States and Spain.

PubMed

Kirillova, Viktorija; Prakas, Petras; Calero-Bernal, Rafael; Gavarāne, Inese; Fernández-García, José Luis; Martínez-González, Manuel; Rudaitytė-Lukošienė, Eglė; Martínez-Estéllez, Miguel Ángel Habela; Butkauskas, Dalius; Kirjušina, Muza

2018-03-12

Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers.

PubMed Central

Reeve, Wayne; O’Hara, Graham; Chain, Patrick; Ardley, Julie; Bräu, Lambert; Nandesena, Kemanthi; Tiwari, Ravi; Copeland, Alex; Nolan, Matt; Han, Cliff; Brettin, Thomas; Land, Miriam; Ovchinikova, Galina; Ivanova, Natalia; Mavromatis, Konstantinos; Markowitz, Victor; Kyrpides, Nikos; Melino, Vanessa; Denton, Matthew; Yates, Ron; Howieson, John

2010-01-01

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp. PMID:21304718

Methylation Pattern of Radish (Raphanus sativus) Nuclear Ribosomal RNA Genes 1

PubMed Central

Delseny, Michel; Laroche, Monique; Penon, Paul

1984-01-01

The methylation pattern of radish Raphanus sativus nuclear rDNA has been investigated using the Hpa II, Msp I, and Hha I restriction enzymes. The presence of numerous target sites for these enzymes has been shown using cloned rDNA fragments. A large fraction of the numerous rDNA units are heavily methylated, being completely resistant to Hpa II and Hpa I. However, specific sites are constantly available in another fraction of the units and are therefore unmethylated. The use of different probes allowed us to demonstrate that hypomethylated sites are present in different regions. Major hypomethylated Hha I sites have been mapped in the 5′ portion of 25S rRNA coding sequence. Among the hypomethylated fraction, different methylation patterns coexist. It has been possible to demonstrate that methylation patterns are specific for particular units. The Hha I pattern of rDNA in tissues of different developmental stages was analyzed. Evidence for possible tissue specific differences in the methylation pattern is reported. Images Fig. 2 Fig. 3 Fig. 5 PMID:16663896

Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions.

PubMed

Harpke, Doerte; Peterson, Angela

2008-05-01

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems.

The Evolution of Ribosomal DNA: Divergent Paralogues and Phylogenetic Implications

PubMed Central

Buckler-IV, E. S.; Ippolito, A.; Holtsford, T. P.

1997-01-01

Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serve as good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies. PMID:9055091

Characterization of the complete chloroplast genome of Platycarya strobilacea (Juglandaceae)

Treesearch

Jing Yan; Kai Han; Shuyun Zeng; Peng Zhao; Keith Woeste; Jianfang Li; Zhan-Lin Liu

2017-01-01

The whole chloroplast genome (cp genome) sequence of Platycarya strobilacea was characterized from Illumina pair-end sequencing data. The complete cp genome was 160,994 bp in length and contained a large single copy region (LSC) of 90,225 bp and a small single copy region (SSC) of 18,371 bp, which were separated by a pair of inverted repeat regions...

Analysis of 16S rDNA and Metagenomic Sequences Revealed Microbial Community and Host-Specific Sequences of Canadian Geese Feces

EPA Science Inventory

There is an increasing concern regarding the public health risks associated with waterfowl fecal pollution as a result of the increase in geese populations (Branta canadensis) in or near U.S. and Canadian recreational waters. Currently, there are no methods that can be used to de...

Hintoniamine, a new calmodulin inhibitor from the endophytic fungal species 39140-2

USDA-ARS?s Scientific Manuscript database

A new fungal endophytic species (39140-2) was isolated from the medicinal plant Hintonia latiflora (Sessé et Mociño ex DC.) Bullock, a Rubiaceae widely used in Mexico as antidiabetic agent. Sequencing of the 28S fraction of the rDNA of this fungal strain and comparing with similar sequences for all ...

Asexual-sexual morph connection in the type species of Berkleasmium.

PubMed

Tanney, Joey; Miller, Andrew N

2017-06-01

Berkleasmium is a polyphyletic genus comprising 37 dematiaceous hyphomycetous species. In this study, independent collections of the type species, B. concinnum , were made from Eastern North America. Nuclear internal transcribed spacer rDNA (ITS) and partial nuc 28S large subunit rDNA (LSU) sequences obtained from collections and subsequent cultures showed that Berkleasmium concinnum is the asexual morph of Neoacanthostigma septoconstrictum ( Tubeufiaceae , Tubeufiales ). Phylogenies inferred from Bayesian inference and maximum likelihood analyses of ITS-LSU sequence data confirmed this asexual-sexual morph connection and a re-examination of fungarium reference specimens also revealed the co-occurrence of N. septoconstrictum ascomata and B. concinnum sporodochia. Neoacanthostigma septoconstrictum is therefore synonymized under B. concinnum on the basis of priority. A specimen identified as N. septoconstrictum from Thailand is described as N. thailandicum sp. nov., based on morphological and genetic distinctiveness.

Chromosomal organization of four classes of repetitive DNA sequences in killifish Orestias ascotanensis Parenti, 1984 (Cyprinodontiformes, Cyprinodontidae)

PubMed Central

Araya-Jaime, Cristian; Lam, Natalia; Pinto, Irma Vila; Méndez, Marco A.; Iturra, Patricia

2017-01-01

Abstract Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. Cytogenetic studies of these species have focused on conventional karyotyping. The aim of this study was to use classical and molecular cytogenetic methods to identify the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNA sequences (histone H3 DNA, U2 snRNA, 18S rDNA and 5S rDNA) in the chromosomes of O. ascotanensis Parenti, 1984, an endemic species restricted to the Salar de Ascotán in the Chilean Altiplano. All individuals analyzed had a diploid number of 48 chromosomes. C-banding identified constitutive heterochromatin mainly in the pericentromeric region of most chromosomes, especially a GC-rich heterochromatic block of the short arm of pair 3. FISH assay with an 18S probe confirmed the location of the NOR in pair 3 and revealed that the minor rDNA cluster occurs interstitially on the long arm of pair 2. Dual FISH identified a single block of U2 snDNA sequences in the pericentromeric regions of a subtelocentric chromosome pair, while histone H3 sites were observed as small signals scattered in throughout the all chromosomes. This work represents the first effort to document the physical organization of the repetitive fraction of the Orestias genome. These data will improve our understanding of the chromosomal evolution of a genus facing serious conservation problems. PMID:29093798

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery

PubMed Central

Mais, Christine; Wright, Jane E.; Prieto, José-Luis; Raggett, Samantha L.; McStay, Brian

2005-01-01

Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein–protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements. PMID:15598984

The Complete Chloroplast and Mitochondrial Genome Sequences of Boea hygrometrica: Insights into the Evolution of Plant Organellar Genomes

PubMed Central

Wang, Xumin; Deng, Xin; Zhang, Xiaowei; Hu, Songnian; Yu, Jun

2012-01-01

The complete nucleotide sequences of the chloroplast (cp) and mitochondrial (mt) genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae) have been determined with the lengths of 153,493 bp and 510,519 bp, respectively. The smaller chloroplast genome contains more genes (147) with a 72% coding sequence, and the larger mitochondrial genome have less genes (65) with a coding faction of 12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of 222 bp, 843 bp, and 1474 bp in length, which divide the genome into a single master circle (MC) and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage. PMID:22291979

Molecular typing of Staphylococcus aureus based on coagulase gene.

PubMed

Javid, Faizan; Taku, Anil; Bhat, Mohd Altaf; Badroo, Gulzar Ahmad; Mudasir, Mir; Sofi, Tanveer Ahmad

2018-04-01

This study was conducted to study the coagulase gene-based genetic diversity of Staphylococcus aureus , isolated from different samples of cattle using restriction fragment length polymorphism (RFLP) and their sequence-based phylogenetic analysis. A total of 192 different samples from mastitic milk, nasal cavity, and pus from skin wounds of cattle from Military Dairy Farm, Jammu, India, were screened for the presence of S. aureus . The presumptive isolates were confirmed by nuc gene-based polymerase chain reaction (PCR). The confirmed S. aureus isolates were subjected to coagulase ( coa ) gene PCR. Different coa genotypes observed were subjected to RFLP using restriction enzymes Hae111 and Alu1 , to obtain the different restriction patterns. One isolate from each restriction pattern was sequenced. These sequences were aligned for maximum homology using the Bioedit softwareandsimilarity in the sequences was inferred with the help of sequence identity matrix. Of 192 different samples,39 (20.31%) isolates of S. aureus were confirmed by targeting nuc gene using PCR. Of 39 S. aureus isolates, 25 (64.10%) isolates carried coa gene. Four different genotypes of coa gene, i.e., 514 bp, 595 bp, 757 bp, and 802 bp were obtained. Two coa genotypes, 595 bp (15 isolates) and 802 bp (4 isolates), were observed in mastitic milk. 514 bp (2 isolates) and 757 bp (4 isolates) coa genotypes were observed from nasal cavity and pus from skin wounds, respectively. On RFLP using both restriction enzymes, four different restriction patterns P1, P2, P3, and P4 were observed. On sequencing, four different sequences having unique restriction patterns were obtained. The most identical sequences with the value of 0.810 were found between isolate S. aureus 514 (nasal cavity) and S. aureus 595 (mastitic milk), and thus, they are most closely related. While as the most distant sequences with the value of 0.483 were found between S. aureus 514 and S. aureus 802 isolates. The study, being localized to only one farm, yielded different RFLP patterns as observed from different sampling sites, which indicates that different S . aureus coagulase typeshave a site-specific predilection. Two coa patterns were observed in mastitic milk indicating multiple origins of infection, with 595 bp coa genotype being predominant in mastitic milk. The coa genotypes and their restriction patterns observed in the present study are novel, not published earlier. 514 and 595 coa variants of S. aureus are genetically most related.

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid

PubMed Central

2011-01-01

Background Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. Results 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Conclusions Paphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants. PMID:21910890

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid.

PubMed

Lan, Tianying; Albert, Victor A

2011-09-12

Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Paphiopedilum species display many chromosomal rearrangements--for example, duplications, translocations, and inversions--but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants.

The cytochrome oxidase subunit I and subunit III genes in Oenothera mitochondria are transcribed from identical promoter sequences

PubMed Central

Hiesel, Rudolf; Schobel, Werner; Schuster, Wolfgang; Brennicke, Axel

1987-01-01

Two loci encoding subunit III of the cytochrome oxidase (COX) in Oenothera mitochondria have been identified from a cDNA library of mitochondrial transcripts. A 657-bp sequence block upstream from the open reading frame is also present in the two copies of the COX subunit I gene and is presumably involved in homologous sequence rearrangement. The proximal points of sequence rearrangements are located 3 bp upstream from the COX I and 1139 bp upstream from the COX III initiation codons. The 5'-termini of both COX I and COX III mRNAs have been mapped in this common sequence confining the promoter region for the Oenothera mitochondrial COX I and COX III genes to the homologous sequence block. ImagesFig. 5. PMID:15981332

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation

PubMed Central

Garcia, S; Kovařík, A

2013-01-01

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S–5.8S–26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S–18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S–5.8S–26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants. PMID:23512008

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation.

PubMed

Garcia, S; Kovařík, A

2013-07-01

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S-5.8S-26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S-18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S-5.8S-26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants.

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana

PubMed Central

Knoll, Alexander; Puchta, Holger

2016-01-01

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline. PMID:27760121

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

PubMed

Röhrig, Sarah; Schröpfer, Susan; Knoll, Alexander; Puchta, Holger

2016-10-01

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

Transposable elements in fish chromosomes: a study in the marine cobia species.

PubMed

Costa, G W W F; Cioffi, M B; Bertollo, L A C; Molina, W F

2013-01-01

Rachycentron canadum, a unique representative of the Rachycentridae family, has been the subject of considerable biotechnological interest due to its potential use in marine fish farming. This species has undergone extensive research concerning the location of genes and multigene families on its chromosomes. Although most of the genome of some organisms is composed of repeated DNA sequences, aspects of the origin and dispersion of these elements are still largely unknown. The physical mapping of repetitive sequences on the chromosomes of R. canadum proved to be relevant for evolutionary and applied purposes. Therefore, here, we present the mapping by fluorescence in situ hybridization of the transposable element (TE) Tol2, the non-LTR retrotransposons Rex1 and Rex3, together with the 18S and 5S rRNA genes in the chromosome of this species. The Tol2 TE, belonging to the family of hAT transposons, is homogeneously distributed in the euchromatic regions of the chromosomes but with huge colocalization with the 18S rDNA sites. The hybridization signals for Rex1 and Rex3 revealed a semi-arbitrary distribution pattern, presenting differentiated dispersion in euchromatic and heterochromatic regions. Rex1 elements are associated preferentially in heterochromatic regions, while Rex3 shows a scarce distribution in the euchromatic regions of the chromosomes. The colocalization of TEs with 18S and 5S rDNA revealed complex chromosomal regions of repetitive sequences. In addition, the nonpreferential distribution of Rex1 and Rex3 in all heterochromatic regions, as well as the preferential distribution of the Tol2 transposon associated with 18S rDNA sequences, reveals a distinct pattern of organization of TEs in the genome of this species. A heterogeneous chromosomal colonization of TEs may confer different evolutionary rates to the heterochromatic regions of this species.

Development of chemiluminescent probe hybridization, RT-PCR and nucleic acid cycle sequencing assays of Sabin type 3 isolates to identify base pair 472 Sabin type 3 mutants associated with vaccine associated paralytic poliomyelitis.

PubMed

Old, M O; Logan, L H; Maldonado, Y A

1997-11-01

Sabin type 3 polio vaccine virus is the most common cause of poliovaccine associated paralytic poliomyelitis. Vaccine associated paralytic poliomyelitis cases have been associated with Sabin type 3 revertants containing a single U to C substitution at bp 472 of Sabin type 3. A rapid method of identification of Sabin type 3 bp 472 mutants is described. An enterovirus group-specific probe for use in a chemiluminescent dot blot hybridization assay was developed to identify enterovirus positive viral lysates. A reverse transcription-polymerase chain reaction (RT-PCR) assay producing a 319 bp PCR product containing the Sabin type 3 bp 472 mutation site was then employed to identify Sabin type 3 isolates. Chemiluminescent nucleic acid cycle sequencing of the purified 319 bp PCR product was then employed to identify nucleic acid sequences at bp 472. The enterovirus group probe hybridization procedure and isolation of the Sabin type 3 PCR product were highly sensitive and specific; nucleic acid cycle sequencing corresponded to the known sequence of stock Sabin type 3 isolates. These methods will be used to identify the Sabin type 3 reversion rate from sequential stool samples of infants obtained after the first and second doses of oral poliovirus vaccine.

Species identification of mutans streptococci by groESL gene sequence.

PubMed

Hung, Wei-Chung; Tsai, Jui-Chang; Hsueh, Po-Ren; Chia, Jean-San; Teng, Lee-Jene

2005-09-01

The near full-length sequences of the groESL genes were determined and analysed among eight reference strains (serotypes a to h) representing five species of mutans group streptococci. The groES sequences from these reference strains revealed that there are two lengths (285 and 288 bp) in the five species. The intergenic spacer between groES and groEL appears to be a unique marker for species, with a variable size (ranging from 111 to 310 bp) and sequence. Phylogenetic analysis of groES and groEL separated the eight serotypes into two major clusters. Strains of serotypes b, c, e and f were highly related and had groES gene sequences of the same length, 288 bp, while strains of serotypes a, d, g and h were also closely related and their groES gene sequence lengths were 285 bp. The groESL sequences in clinical isolates of three serotypes of S. mutans were analysed for intraspecies polymorphism. The results showed that the groESL sequences could provide information for differentiation among species, but were unable to distinguish serotypes of the same species. Based on the determined sequences, a PCR assay was developed that could differentiate members of the mutans streptococci by amplicon size and provide an alternative way for distinguishing mutans streptococci from other viridans streptococci.

Accurate, Rapid Taxonomic Classification of Fungal Large-Subunit rRNA Genes

PubMed Central

Liu, Kuan-Liang; Porras-Alfaro, Andrea; Eichorst, Stephanie A.

2012-01-01

Taxonomic and phylogenetic fingerprinting based on sequence analysis of gene fragments from the large-subunit rRNA (LSU) gene or the internal transcribed spacer (ITS) region is becoming an integral part of fungal classification. The lack of an accurate and robust classification tool trained by a validated sequence database for taxonomic placement of fungal LSU genes is a severe limitation in taxonomic analysis of fungal isolates or large data sets obtained from environmental surveys. Using a hand-curated set of 8,506 fungal LSU gene fragments, we determined the performance characteristics of a naïve Bayesian classifier across multiple taxonomic levels and compared the classifier performance to that of a sequence similarity-based (BLASTN) approach. The naïve Bayesian classifier was computationally more rapid (>460-fold with our system) than the BLASTN approach, and it provided equal or superior classification accuracy. Classifier accuracies were compared using sequence fragments of 100 bp and 400 bp and two different PCR primer anchor points to mimic sequence read lengths commonly obtained using current high-throughput sequencing technologies. Accuracy was higher with 400-bp sequence reads than with 100-bp reads. It was also significantly affected by sequence location across the 1,400-bp test region. The highest accuracy was obtained across either the D1 or D2 variable region. The naïve Bayesian classifier provides an effective and rapid means to classify fungal LSU sequences from large environmental surveys. The training set and tool are publicly available through the Ribosomal Database Project (http://rdp.cme.msu.edu/classifier/classifier.jsp). PMID:22194300

Genetic characteristics of Streptococcus dysgalactiae isolated from cage cultured cobia, Rachycentron canadum (L.).

PubMed

Tsai, M-A; Wang, P-C; Yoshida, T; Chen, S-C

2015-12-01

Disease outbreaks occurred during 2007-2013 in Taiwan with 2.5-10% mortality among the cage cultured cobia, Rachycentron canadum (L.), characterized by the presence of polyserositis, pericarditis and peritonitis. The micro-organisms isolated from internal organs were Gram-positive cocci. The isolates were confirmed as Streptococcus dysgalactiae by a polymerase chain reaction assay that yielded the expected specific 259 bp amplicon. Additionally, partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish was also compared and produced 100% sequence identity with S. dysgalactiae (GenBank accession number AB252398). The genetic characterization was then determined by pulsed-field gel electrophoresis (PFGE) analysis. Based on PFGE, the Apa I or Sma I digestion patterns of chromosomal DNA of these isolates were grouped into three main clusters. Taiwanese strains were divided into two clusters, and the tet(M) gene was detected in cluster 1 (pulsotypes: A1-A2 and S1-S3), but not in cluster 2 strains (pulsotypes: A3-A4 and S4-S5). Three Japanese strains from amberjack, Seriola dumerili (Risso), were grouped into cluster 3 (pulsotypes: A5-A7 and S6-S8) and displayed no mortality to cobia in the challenge experiment. Conversely, Taiwanese strains from cobia and snubnose pompano, Trachinotus blochii (L.), displayed a mortality rate of 50-87.5% in cobia. © 2014 John Wiley & Sons Ltd.

Isolation of Raoultella planticola from refillable antimicrobial liquid soap dispensers in a dental setting

PubMed Central

Tomlin, Nancy; Ruby, John D.

2014-01-01

Background Liquid antimicrobial soaps are commonly used in the dental healthcare setting for hand washing to minimize the potential spread of infectious agents to healthcare workers (HCW) and patients. The purpose of the current study was to evaluate possible bacterial contamination of antimicrobial liquid soap dispensers located in two institutional comprehensive dental care clinics. Methods Fourteen soap dispensers and original stock containers were sampled. A 1 ml aliquot was diluted in 10 ml of phosphate buffer (Tween 80). Serial dilutions were plated in duplicate on neutralizing agar and incubated for 7 days. Molecular identification was performed using 500 bp comparisons of 16S rDNA sequencing. Taq PCR was performed with sequence specific primers for Raoultella species. Results Bacterial growth was observed at 18 hours for 57% (8/14) of soap dispenser samples. Bacterial densities ranged from 4 ×102–6 ×109 CFU/mL. Original commercial containers exhibited no growth. Isolates were identified as Raoultella (Klebsiella) planticola. Conclusions This is the first study indicating recovery of R. planticola from antimicrobial liquid soap dispensers. R. planticola is a recognized environmental opportunistic pathogen that potentially poses a health concern. Practical Implications These findings indicate compliance problems with infection prevention recommendations and support the CDC’s recommendation that dispensers should not be “topped off”. High bacterial loads of R. planticola are inconsistent with infection control practices and are a concern since transmission and possible infection to the HCW or the patient may occur. PMID:25819655

Cross-fostering immediately after birth induces a permanent microbiota shift that is shaped by the nursing mother.

PubMed

Daft, Joseph G; Ptacek, Travis; Kumar, Ranjit; Morrow, Casey; Lorenz, Robin G

2015-01-01

Current research has led to the appreciation that there are differences in the commensal microbiota between healthy individuals and individuals that are predisposed to disease. Treatments to reverse disease pathogenesis through the manipulation of the gastrointestinal (GI) microbiota are now being explored. Normalizing microbiota between different strains of mice in the same study is also needed to better understand disease pathogenesis. Current approaches require repeated delivery of bacteria and large numbers of animals and vary in treatment start time. A method is needed that can shift the microbiota of predisposed individuals to a healthy microbiota at an early age and sustain this shift through the lifetime of the individual. We tested cross-fostering of pups within 48 h of birth as a means to permanently shift the microbiota from birth. Taxonomical analysis revealed that the nursing mother was the critical factor in determining bacterial colonization, instead of the birth mother. Data was evaluated using bacterial 16S rDNA sequences from fecal pellets and sequencing was performed on an Illumina Miseq using a 251 bp paired-end library. The results show that cross-fostering is an effective means to induce an early and maintained shift in the commensal microbiota. This will allow for the evaluation of a prolonged microbial shift and its effects on disease pathogenesis. Cross-fostering will also eliminate variation within control models by normalizing the commensal microbiota between different strains of mice.

Mitochondrial genome sequences of landsnails Aegista diversifamilia and Dolicheulota formosensis (Gastropoda: Pulmonata: Stylommatophora).

PubMed

Huang, Chih-Wei; Lin, Si-Min; Wu, Wen-Lung

2016-07-01

The first mitochondrial genome sequences of Aegista and Dolicheulota belonging to Bradybaenidae are described in this report. Mitogenomic sequences were generated from Illumina paired-end sequencing. The complete mitogenome of Aegista diversifamilia was 14,039 bp in length and nearly complete mitogenome of Dolicheulota formosensis was 14,237 bp. Both mitogenomes consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. Most genes were overlapped with neighboring genes that the overlapping regions ranged from 2 to 64 bp in A. diversifamilia and from 1 to 45 bp in D. formosensis. Novel gene arrangement, tRNA-Tyr-ND3-tRNA-Trp, was identified in A. diversifamilia, whereas D. formosensis showed identical gene order to other Bradybaenidae mitogenomes. Maximum likelihood phylogenetic tree suggested Aegista as a sister clade to Euhadra and Dolicheulota. Bradybaenidae is monophyly sister clade to Camaenidae.

The complete chloroplast genome sequence of Dendrobium officinale.

PubMed

Yang, Pei; Zhou, Hong; Qian, Jun; Xu, Haibin; Shao, Qingsong; Li, Yonghua; Yao, Hui

2016-01-01

The complete chloroplast sequence of Dendrobium officinale, an endangered and economically important traditional Chinese medicine, was reported and characterized. The genome size is 152,018 bp, with 37.5% GC content. A pair of inverted repeats (IRs) of 26,284 bp are separated by a large single-copy region (LSC, 84,944 bp) and a small single-copy region (SSC, 14,506 bp). The complete cp DNA contains 83 protein-coding genes, 39 tRNA genes and 8 rRNA genes. Fourteen genes contained one or two introns.

First complete genome sequence of infectious laryngotracheitis virus

PubMed Central

2011-01-01

Background Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. To date, only one complete genomic sequence of ILTV has been reported. This sequence was generated by concatenating partial sequences from six different ILTV strains. Thus, the full genomic sequence of a single (individual) strain of ILTV has not been determined previously. This study aimed to use high throughput sequencing technology to determine the complete genomic sequence of a live attenuated vaccine strain of ILTV. Results The complete genomic sequence of the Serva vaccine strain of ILTV was determined, annotated and compared to the concatenated ILTV reference sequence. The genome size of the Serva strain was 152,628 bp, with a G + C content of 48%. A total of 80 predicted open reading frames were identified. The Serva strain had 96.5% DNA sequence identity with the concatenated ILTV sequence. Notably, the concatenated ILTV sequence was found to lack four large regions of sequence, including 528 bp and 594 bp of sequence in the UL29 and UL36 genes, respectively, and two copies of a 1,563 bp sequence in the repeat regions. Considerable differences in the size of the predicted translation products of 4 other genes (UL54, UL30, UL37 and UL38) were also identified. More than 530 single-nucleotide polymorphisms (SNPs) were identified. Most SNPs were located within three genomic regions, corresponding to sequence from the SA-2 ILTV vaccine strain in the concatenated ILTV sequence. Conclusions This is the first complete genomic sequence of an individual ILTV strain. This sequence will facilitate future comparative genomic studies of ILTV by providing an appropriate reference sequence for the sequence analysis of other ILTV strains. PMID:21501528

EvaGreen real-time PCR protocol for specific 'Candidatus Phytoplasma mali' detection and quantification in insects.

PubMed

Monti, Monia; Martini, Marta; Tedeschi, Rosemarie

2013-01-01

In this paper the validation and implementation of a Real-time PCR protocol based on ribosomal protein genes has been carried out for sensitive and specific quantification of 'Candidatus (Ca.) Phytoplasma mali' (apple proliferation phytoplasma, APP) in insects. The method combines the use of EvaGreen(®) dye as chemistry detection system and the specific primer pair rpAP15f-mod/rpAP15r3, which amplifies a fragment of 238 bp of the ribosomal protein rplV (rpl22) gene of APP. Primers specificity was demonstrated by running in the same Real-time PCR 'Ca. Phytoplasma mali' samples with phytoplasmas belonging to the same group (16SrX) as 'Ca. Phytoplasma pyri' and 'Ca. Phytoplasma prunorum', and also phytoplasmas from different groups, as 'Ca. Phytoplasma phoenicium' (16SrIX) and Flavescence dorée phytoplasma (16SrV). 'Ca. Phytoplasma mali' titre in insects was quantified using a specific approach, which relates the concentration of the phytoplasma to insect 18S rDNA. Absolute quantification of APP and insect 18S rDNA were calculated using standard curves prepared from serial dilutions of plasmids containing rplV-rpsC and a portion of 18S rDNA genes, respectively. APP titre in insects was expressed as genome units (GU) of phytoplasma per picogram (pg) of individual insect 18S rDNA. 'Ca. Phytoplasma mali' concentration in examined samples (Cacopsylla melanoneura overwintered adults) ranged from 5.94 × 10(2) to 2.51 × 10(4) GU/pg of insect 18S rDNA. Repeatability and reproducibility of the method were also evaluated by calculation of the coefficient of variation (CV%) of GU of phytoplasma and pg of 18S rDNA fragment for both assays. CV less than 14% and 9% (for reproducibility test) and less than 10 and 11% (for repeatability test) were obtained for phytoplasma and insect qPCR assays, respectively. Sensitivity of the method was also evaluated, in comparison with conventional 16S rDNA-based nested-PCR procedure. The method described has been demonstrated reliable, sensitive and specific for the quantification of 'Ca. Phytoplasma mali' in insects. The possibility to study the trend of phytoplasma titre in the vectors will allow a deepen investigation on the epidemiology of the disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes.

PubMed

Ewing, Aren; Brubaker, Shane; Somanchi, Aravind; Yu, Esther; Rudenko, George; Reyes, Nina; Espina, Karen; Grossman, Arthur; Franklin, Scott

2014-08-01

Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships.

16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes1

PubMed Central

Ewing, Aren; Brubaker, Shane; Somanchi, Aravind; Yu, Esther; Rudenko, George; Reyes, Nina; Espina, Karen; Grossman, Arthur; Franklin, Scott

2014-01-01

Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships. PMID:25937672

Ribosomal DNA identification of Nosema/Vairimorpha in freshwater polychaete, Manayunkia speciosa, from Oregon/California and the Laurentian Great Lakes

USGS Publications Warehouse

Malakauskas, David M.; Altman, Emory C.; Malakauskas, Sarah J.; Thiem, Suzanne M.; Schloesser, Donald W.

2015-01-01

We examined Manayunkia speciosa individuals from the Klamath River, Oregon/California and Lake Erie, Michigan, USA for the presence of Microsporidia. We identified microsporidian spores and sequenced their SSU, ITS, and part of the LSU rDNA. Phylogenetic analysis of SSU rDNA indicated spores from both populations belonged to the Nosema/Vairimorpha clade. PCR showed an infection prevalence in Lake Erie M. speciosa of 0.6% (95% CI = 0.5%, 0.7%). This represents the first known example of molecularly characterized Nosema/Vairimorpha isolates infecting a non-arthropod host.

Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla.

PubMed

Zhang, Mengyan; Wang, Siyao; Yin, Jing; Li, Chunxiao; Zhan, Yaguang; Xiao, Jialei; Liang, Tian; Li, Xin

2016-09-01

Betula platyphylla is a rich repository of pharmacologically active secondary metabolites known as birch triterpenoids (TBP). Here, we cloned the squalene synthase (SS) and squalene epoxidase genetic (SE) sequences from B. platyphylla that encode the key enzymes that are involved in triterpenoid biosynthesis and analyzed the conserved domains and phylogenetics of their corresponding proteins. The full-length sequence of BpSS is 1588 bp with a poly-A tail, which contained an open reading frame (ORF) of 1241 bp that encoded a protein of 413 amino acids. Additionally, the BpSE full-length sequence of 2040 bp with a poly-A tail was also obtained, which contained an ORF of 1581 bp encoding a protein of 526 amino acids. Their organ-specific expression patterns in 4-week-old tissue culture seedlings of B. platyphylla were detected by real-time PCR and showed that they were all highly expressed in leaves, as compared to stem and root tissues. Additionaly, both BpSS and BpSE were enhanced following stimulation with ethephon and MeJA. The expression of BpSS was enhanced by ABA, whereas BpSE was not. The SA treatment did not affect the BpSS and BpSE transcripts notably. Using a genome walking approach, promoter sequences of 965 and 1193 bp, respectively, for BpSS and BpSE were isolated, and they revealed several key cis-regulatory elements known to be involved in the response to phytohormone and abiotic plant stress. We also found that the BpSS protein is localized in the cytoplasm. Opening reading frames of BpSS and BpSE were ligated into yeast expression plasmid pYES2 under control of GAL1 promoter and introduced into the yeast INVScl1 strain. The transformants were cultured for 12 h, the squalene content of galactose-induced BpSS expression yeast cells was 13.2 times of control (empty vector control yeast cells) by high-performance liquid chromatography (HPLC) test method. And, the squalene epoxidase activity of induced BpSE expression yeast cell was about 11.8 times of control. These indicated that we cloned birch BpSS and BpSE that were indeed involved in the synthesis of triteropenoids. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the triterpenoids biosynthesis of B. platyphylla. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the biosynthesis of birch triterpenoids.

Multigene assessment of the species boundaries and sexual status of the basidiomycetous yeasts Cryptococcus flavescens and C. terrestris (Tremellales).

PubMed

Yurkov, Andrey; Guerreiro, Marco A; Sharma, Lav; Carvalho, Cláudia; Fonseca, Álvaro

2015-01-01

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful.

Next-Generation Sequencing Reveals Significant Bacterial Diversity of Botrytized Wine

PubMed Central

Bokulich, Nicholas A.; Joseph, C. M. Lucy; Allen, Greg; Benson, Andrew K.; Mills, David A.

2012-01-01

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies. PMID:22563494

Multigene Assessment of the Species Boundaries and Sexual Status of the Basidiomycetous Yeasts Cryptococcus flavescens and C. terrestris (Tremellales)

PubMed Central

Sharma, Lav; Carvalho, Cláudia; Fonseca, Álvaro

2015-01-01

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful. PMID:25811603

Mycobacterium tuberculosis Infection of Domesticated Asian Elephants, Thailand

PubMed Central

Angkawanish, Taweepoke; Sirimalaisuwan, Anucha; Kaewsakhorn, Thattawan; Boonsri, Kittikorn; Rutten, Victor P.M.G.

2010-01-01

Four Asian elephants were confirmed to be infected with Mycobacterium tuberculosis by bacterial culture, other diagnostic procedures, and sequencing of 16S–23S rDNA internal transcribed spacer region, 16S rRNA, and gyrase B gene sequences. Genotyping showed that the infectious agents originated from 4 sources in Thailand. To identify infections, a combination of diagnostic assays is essential. PMID:21122228

[Variability of nuclear 18S-25S rDNA of Gentiana lutea L. in nature and in tissue culture in vitro].

PubMed

Mel'nyk, V M; Spiridonova, K V; Andrieiev, I O; Strashniuk, N M; Kunakh, V A

2004-01-01

18S-25S rDNA sequence in genomes of G. lutea plants from different natural populations and from tissue culture has been studied with blot-hybridization method. It was shown that ribosomal repeats are represented by the variants which differ for their size and for the presence of additional HindIII restriction site. Genome of individual plant usually possesses several variants of DNA repeats. Interpopulation variability according to their quantitative ratio and to the presence of some of them has been shown. Modifications of the range of rDNA repeats not exceeding intraspecific variability were observed in callus tissues in comparison with the plants of initial population. Non-randomness of genome modifications in the course of cell adaptation to in vitro conditions makes it possible to some extent to forecast these modifications in tissue culture.

Possibilities in identification of genomic species of Burkholderia cepacia complex by PCR and RFLP.

PubMed

Navrátilová, Lucie; Chromá, Magdalena; Hanulík, Vojtech; Raclavský, Vladislav

2013-01-01

The strains belonging to Burkholderia cepacia complex are important opportunistic pathogens in immunocompromised patients and cause serious diseases. It is possible to obtain isolates from soil, water, plants and human samples. Taxonomy of this group is difficult. Burkholderia cepacia complex consists of seventeen genomic species and the genetic scheme is based on recA gene. Commonly, first five genomovars occurre in humans, mostly genomovars II and III, subdivision IIIA. Within this study we tested identification of first five genomovars by PCR with following melting analysis and RFLP. The experiments were targeted on eubacterial 16S rDNA and specific gene recA, which allowed identification of all five genomovars. RecA gene appeared as more suitable than 16S rDNA, which enabled direct identification of only genomovars II and V; genomovars I, III and IV were similar within 16S rDNA sequence.

Phylogeny of five species of Nusuttodinium gen. nov. (Dinophyceae), a genus of unarmoured kleptoplastidic dinoflagellates.

PubMed

Takano, Yoshihito; Yamaguchi, Haruyo; Inouye, Isao; Moestrup, Øjvind; Horiguchi, Takeo

2014-12-01

Cells of five unarmoured kleptoplastidic dinoflagellates, Amphidinium latum, Amphidinium poecilochroum, Gymnodinium amphidinioides, Gymnodinium acidotum and Gymnodinium aeruginosum were observed under light and/or scanning electron microscopy and subjected to single-cell PCR. The SSU rDNA and the partial LSU rDNA of all the examined species were sequenced, and the SSU rDNA of G. myriopyrenoides was sequenced. Phylogenetic analyses revealed that the unarmoured kleptoplastidic species formed a monophyletic clade within the Gymnodinium-clade sensu Daugbjerg et al. (2000). The sister taxa for this clade were Gymnodinium palustre and Spiniferodinium galeiforme, both of which possess brown-coloured chloroplasts. The results indicated that acquisition of kleptoplastidy in these unarmoured dinoflagellates was a single event and that these unarmoured kleptoplastidic dinoflagellates may have evolved from a form with permanent chloroplasts. Molecular trees suggested that the acquisition of kleptoplastidy took place in a marine habitat and later some species colonized the freshwater habitat. Because these unarmoured kleptoplastidic dinoflagellates are monophyletic and characterized by distinct morphological and cytological features (including the presence of the same type of apical groove, absence of nuclear chambers in the nuclear envelope, absence of genuine chloroplasts, and the possession of kleptochloroplasts), we propose the establishment of a new genus, Nusuttodinium, to accommodate all these dinoflagellates. Copyright © 2014 Elsevier GmbH. All rights reserved.

Probiotic Candidates from Fish Pond Water in Central Java Indonesia

NASA Astrophysics Data System (ADS)

Harjuno Condro Haditomo, Alfabetian; Desrina; Sarjito; Budi Prayitno, S.

2018-02-01

Aeromonas hydrophilla is a major bacterial pathogen of intensive fresh water fish culture in Indonesia. An alternative method to control the pathogen is using probiotics. Probiotics is usually consist of live microorganisms which when administered in adequate amounts confer a health benefits on host. The aim of this research was to determine the probiotic candidates against A. hydrophilla which identified based on the 16S rDNA gene sequences. This research was started with field survey to obtained the probiotic candidate and continue with laboratory experiment. Probiotic candidates were isolated from fish pond water located in Boyolali, and Banjarnegara Regency, Central Java, Indonesia. A total of 133 isolates bacteria were isolated and cultured on to TSA, TSB and GSP medium. Out of 133 isolates only 30 isolates showed inhibition to A.hydrophilla activity. Three promising isolates were identified with PCR using primer for 16S rDNA. Based on 16S rDNA sequence analysis, all three isolates were belong to Bacillus genus. Isolate CKlA21, CKlA28, and CBA14 respectively were closely related to Bacillus sp. 13843 (GenBank accession no. JN874760.1 -100% homology), Bacillus subtilis strain H13 (GenBank accession no.KT907045.1 -- 99% homology), and Bacillus sp. strain 22-4 (GenBank accession no. KX816417.1 -- 97% homology).

Asymmetric Epigenetic Modification and Elimination of rDNA Sequences by Polyploidization in Wheat[W

PubMed Central

Guo, Xiang

2014-01-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. PMID:25415973

Endophytic bacteria in plant tissue culture: differences between easy- and difficult-to-propagate Prunus avium genotypes.

PubMed

Quambusch, Mona; Pirttilä, Anna Maria; Tejesvi, Mysore V; Winkelmann, Traud; Bartsch, Melanie

2014-05-01

The endophytic bacterial communities of six Prunus avium L. genotypes differing in their growth patterns during in vitro propagation were identified by culture-dependent and culture-independent methods. Five morphologically distinct isolates from tissue culture material were identified by 16S rDNA sequence analysis. To detect and analyze the uncultivable fraction of endophytic bacteria, a clone library was established from the amplified 16S rDNA of total plant extract. Bacterial diversity within the clone libraries was analyzed by amplified ribosomal rDNA restriction analysis and by sequencing a clone for each identified operational taxonomic unit. The most abundant bacterial group was Mycobacterium sp., which was identified in the clone libraries of all analyzed Prunus genotypes. Other dominant bacterial genera identified in the easy-to-propagate genotypes were Rhodopseudomonas sp. and Microbacterium sp. Thus, the community structures in the easy- and difficult-to-propagate cherry genotypes differed significantly. The bacterial genera, which were previously reported to have plant growth-promoting effects, were detected only in genotypes with high propagation success, indicating a possible positive impact of these bacteria on in vitro propagation of P. avium, which was proven in an inoculation experiment. © The Author 2014. Published by Oxford University Press. All rights reserved.

Asymmetric epigenetic modification and elimination of rDNA sequences by polyploidization in wheat.

PubMed

Guo, Xiang; Han, Fangpu

2014-11-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. © 2014 American Society of Plant Biologists. All rights reserved.

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea.

PubMed

Sun, Wei; Dai, Shikun; Jiang, Shumei; Wang, Guanghua; Liu, Guohui; Wu, Houbo; Li, Xiang

2010-06-01

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group

NASA Technical Reports Server (NTRS)

La Duc, Myron T.; Satomi, Masataka; Agata, Norio; Venkateswaran, Kasthuri

2004-01-01

Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.

Intra-isolate genome variation in arbuscular mycorrhizal fungi persists in the transcriptome.

PubMed

Boon, E; Zimmerman, E; Lang, B F; Hijri, M

2010-07-01

Arbuscular mycorrhizal fungi (AMF) are heterokaryotes with an unusual genetic makeup. Substantial genetic variation occurs among nuclei within a single mycelium or isolate. AMF reproduce through spores that contain varying fractions of this heterogeneous population of nuclei. It is not clear whether this genetic variation on the genome level actually contributes to the AMF phenotype. To investigate the extent to which polymorphisms in nuclear genes are transcribed, we analysed the intra-isolate genomic and cDNA sequence variation of two genes, the large subunit ribosomal RNA (LSU rDNA) of Glomus sp. DAOM-197198 (previously known as G. intraradices) and the POL1-like sequence (PLS) of Glomus etunicatum. For both genes, we find high sequence variation at the genome and transcriptome level. Reconstruction of LSU rDNA secondary structure shows that all variants are functional. Patterns of PLS sequence polymorphism indicate that there is one functional gene copy, PLS2, which is preferentially transcribed, and one gene copy, PLS1, which is a pseudogene. This is the first study that investigates AMF intra-isolate variation at the transcriptome level. In conclusion, it is possible that, in AMF, multiple nuclear genomes contribute to a single phenotype.

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov

NASA Technical Reports Server (NTRS)

Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.; Deinhard, G.; Poralla, K.

1992-01-01

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs.

PubMed

Yuan, Xiao-Long; Zhang, Zhe; Pan, Rong-Yang; Gao, Ning; Deng, Xi; Li, Bin; Zhang, Hao; Sangild, Per Torp; Li, Jia-Qi

2017-01-01

Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome. We found that the Msp I-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110-220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40-110 bp, 110-220 bp and 40-220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110-220 bp fragment size appeared to be deeper than in the 40-110 bp and 40-220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions. In this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.

Phylogenetic Relationships of Cucullanidae (Nematoda), with Observations on Seuratoidea and the Monophyly of Cucullanus, Dichelyne and Truttaedacnitis.

PubMed

Choudhury, Anindo; Nadler, Steven A

2016-02-01

The phylogenetic relationships of Cucullanidae were explored using near-complete sequences of the 18S rDNA (rRNA gene). Sequences (1,750-1,760 bp) were obtained from 7 species of Cucullanidae belonging to 3 genera, Cucullanus (2 spp.), Dichelyne (2 spp.), Truttaedacnitis (3 spp.), and 1 species of Quimperiidae ( Paraseuratum sp.). These sequences were aligned with those of 128 other nematode species available in GenBank, including 3 other cucullanids (Dichelyne mexicanus, Cucullanus robustus, and Cucullanus baylisi) and 2 non-cucullanid seuratoids (Paraquimperia africana, and Linstowinema sp.). Bayesian (BPP) and maximum likelihood (ML) analyses of 2 different datasets strongly supported a monophyletic Cucullanidae. Bayesian analysis placed this family as the sister group to a clade containing species of Diplogasterida, Strongylida, Rhabditida, and Tylenchida with very strong support. Neither BPP nor ML analyses recovered a close relationship of Cucullanidae to Ascaridida. None of the 3 non-cucullanid seuratoid species were sister to Cucullanidae, nor did they form a monophyletic group of their own, which questions the monophyly of Seuratoidea and the relationships among species within this superfamily. The 3 genera of cucullanids were also not monophyletic, although morphologically similar species such as the 2 species of Cucullanus from Neotropical catfishes and 2 species of Dichelyne from Nearctic ictalurid catfishes were sister taxa with strong support. The results were ambiguous with respect to the relationship of 2 Truttaedacnitis spp. in Nearctic freshwater fishes but do not support Truttaedacnitis heterodonti, a parasite of heterodontid sharks, as belonging to this genus. The study shows that all aspects of the conventional classification of Seuratoidea and its taxa should be scrutinized by even more extensive sampling across hosts and habitats.

Tenebrio molitor antifreeze protein gene identification and regulation.

PubMed

Qin, Wensheng; Walker, Virginia K

2006-02-15

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum)

PubMed Central

Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

2015-01-01

We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes—rpoC2, ycf3, accD, and clpP—have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum. PMID:25966355

bpRNA: large-scale automated annotation and analysis of RNA secondary structure.

PubMed

Danaee, Padideh; Rouches, Mason; Wiley, Michelle; Deng, Dezhong; Huang, Liang; Hendrix, David

2018-05-09

While RNA secondary structure prediction from sequence data has made remarkable progress, there is a need for improved strategies for annotating the features of RNA secondary structures. Here, we present bpRNA, a novel annotation tool capable of parsing RNA structures, including complex pseudoknot-containing RNAs, to yield an objective, precise, compact, unambiguous, easily-interpretable description of all loops, stems, and pseudoknots, along with the positions, sequence, and flanking base pairs of each such structural feature. We also introduce several new informative representations of RNA structure types to improve structure visualization and interpretation. We have further used bpRNA to generate a web-accessible meta-database, 'bpRNA-1m', of over 100 000 single-molecule, known secondary structures; this is both more fully and accurately annotated and over 20-times larger than existing databases. We use a subset of the database with highly similar (≥90% identical) sequences filtered out to report on statistical trends in sequence, flanking base pairs, and length. Both the bpRNA method and the bpRNA-1m database will be valuable resources both for specific analysis of individual RNA molecules and large-scale analyses such as are useful for updating RNA energy parameters for computational thermodynamic predictions, improving machine learning models for structure prediction, and for benchmarking structure-prediction algorithms.

Bioinformatic analysis of phage AB3, a phiKMV-like virus infecting Acinetobacter baumannii.

PubMed

Zhang, J; Liu, X; Li, X-J

2015-01-16

The phages of Acinetobacter baumannii has drawn increasing attention because of the multi-drug resistance of A. baumanni. The aim of this study was to sequence Acinetobacter baumannii phage AB3 and conduct bioinformatic analysis to lay a foundation for genome remodeling and phage therapy. We isolated and sequenced A. baumannii phage AB3 and attempted to annotate and analyze its genome. The results showed that the genome is a double-stranded DNA with a total length of 31,185 base pairs (bp) and 97 open reading frames greater than 100 bp. The genome includes 28 predicted genes, of which 24 are homologous to phage AB1. The entire coding sequence is located on the negative strand, representing 90.8% of the total length. The G+C mol% was 39.18%, without areas of high G+C content over 200 bp in length. No GC island, tRNA gene, or repeated sequence was identified. Gene lengths were 120-3099 bp, with an average of 1011 bp. Six genes were found to be greater than 2000 bp in length. Genomic alignment and phylogenetic analysis of the RNA polymerase gene showed that similar to phage AB1, phage AB3 is a phiKMV-like virus in the T7 phage family.

Two new species of the genus Candida in the Zygoascus clade, Candida lundiana sp. nov. and Candida suthepensis sp. nov., isolated from raw honey in Thailand.

PubMed

Saksinchai, Sujinan; Suzuki, Motofumi; Lumyong, Saisamorn; Ohkuma, Moriya; Chantawannakul, Panuwan

2012-03-01

During a survey of yeasts associated with raw honey collected in Thailand, two strains of the Zygoascus clade were isolated from the Asian cavity-nesting honeybee Apis cerana and the stingless bee Homotrigona fimbriata. Phylogeny based on 26S rDNA D1/D2 sequences placed these yeasts as members of a clade including Candida bituminiphila, Candida patagonica and Candida polysorbophila. The strains of the two novel species, CBS 12271(T) and CBS 12270(T), respectively, could be unquestionably distinguished from their relatives by rDNA sequences and other taxonomic characteristics. Therefore, the novel anamorphic species, Candida lundiana sp. nov. (type strain CBS 12271(T) = JCM 16823(T)) and Candida suthepensis sp. nov. (type strain CBS 12270(T) = JCM 16822(T)) are described.

A century of paraphyly: a molecular phylogeny of katydids (Orthoptera: Tettigoniidae) supports multiple origins of leaf-like wings.

PubMed

Mugleston, Joseph D; Song, Hojun; Whiting, Michael F

2013-12-01

The phylogenetic relationships of Tettigoniidae (katydids and bush-crickets) were inferred using molecular sequence data. Six genes (18S rDNA, 28S rDNA, Cytochrome Oxidase II, Histone 3, Tubulin Alpha I, and Wingless) were sequenced for 135 ingroup taxa representing 16 of the 19 extant katydid subfamilies. Five subfamilies (Tettigoniinae, Pseudophyllinae, Mecopodinae, Meconematinae, and Listroscelidinae) were found to be paraphyletic under various tree reconstruction methods (Maximum Likelihood, Bayesisan Inference and Maximum Parsimony). Seven subfamilies - Conocephalinae, Hetrodinae, Hexacentrinae, Saginae, Phaneropterinae, Phyllophorinae, and Lipotactinae - were each recovered as well-supported monophyletic groups. We mapped the small and exposed thoracic auditory spiracle (a defining character of the subfamily Pseudophyllinae) and found it to be homoplasious. We also found the leaf-like wings of katydids have been derived independently in at least six lineages. Copyright © 2013 Elsevier Inc. All rights reserved.

Use of rDNA polymorphism for identification of Heterophyidae infecting freshwater fishes.

PubMed

Dzikowski, R; Levy, M G; Poore, M F; Flowers, J R; Paperna, I

2004-04-21

Infections by trematodes are among the most common fish-borne zoonoses. Metacercariae of the Family Heterophyidae in marine and freshwater fishes are nonfastidious in their choice of definitive hosts, and therefore, cause infections in human and domestic animals. In the present study, species-specific polymerase chain reaction (PCR) assays were developed for identifying and differentiating the various species examined. Sequencing and aligning the 18S (SSU) rDNA revealed interspecific variation for which species-specific DNA oligonucleotides were designed and used for the identification of 6 heterophyid species recovered from piscivorous birds. The oligonucleotides were further used to evaluate the various stages (cercariae recovered from snails, metacercariae recovered from fish and adult trematodes) of the digeneans. By applying this method we elucidated for the first time the life cycle of Pygidiopsis genata. The phylogenetic interrelationship among the newly sequenced species of Heterophyidae is outlined.

IM-TORNADO: a tool for comparison of 16S reads from paired-end libraries.

PubMed

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.

Genetic assemblage of Sarcocystis spp. in Malaysian snakes

PubMed Central

2013-01-01

Background Sarcocystis species are protozoan parasites with a wide host range including snakes. Although there were several reports of Sarcocytis species in snakes, their distribution and prevalence are still not fully explored. Methods In this study, fecal specimens of several snake species in Malaysia were examined for the presence of Sarcocystis by PCR of 18S rDNA sequence. Microscopy examination of the fecal specimens for sporocysts was not carried as it was difficult to determine the species of the infecting Sarcocystis. Results Of the 28 snake fecal specimens, 7 were positive by PCR. BLASTn and phylogenetic analyses of the amplified 18S rDNA sequences revealed the snakes were infected with either S. nesbitti, S. singaporensis, S. zuoi or undefined Sarcocystis species. Conclusion This study is the first to report Sarcocystis infection in a cobra, and S. nesbitti in a reticulated python. PMID:24010903

Length and sequence variability in mitochondrial control region of the milkfish, Chanos chanos.

PubMed

Ravago, Rachel G; Monje, Virginia D; Juinio-Meñez, Marie Antonette

2002-01-01

Extensive length variability was observed in the mitochondrial control region of the milkfish, Chanos chanos. The nucleotide sequence of the control region and flanking regions was determined. Length variability and heteroplasmy was due to the presence of varying numbers of a 41-bp tandemly repeated sequence and a 48-bp insertion/deletion (indel). The structure and organization of the milkfish control region is similar to that of other teleost fish and vertebrates. However, extensive variation in the copy number of tandem repeats (4-20 copies) and the presence of a relatively large (48-bp) indel, are apparently uncommon in teleost fish control region sequences reported to date. High sequence variability of control region peripheral domains indicates the potential utility of selected regions as markers for population-level studies.

The phylogenetic position of an Armillaria species from Amami-Oshima, a subtropical island of Japan, based on elongation factor and ITS sequences

Treesearch

Yuko Ota; Mee-Sook Kim; Hitoshi Neda; Ned B. Klopfenstein; Eri Hasegawa

2011-01-01

An undetermined Armillaria species was collected on Amami-Oshima, a subtropical island of Japan. The phylogenetic position of the Armillaria sp. was determined using sequences of the elongation factor-1a (EF-1a) gene and the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The phylogenetic analyses based on EF-1a and ITS sequences...

The morphological and molecular identity of Longidorus piceicola Lišková, Robbins & Brown, 1997 from Romania (Nematoda, Dorylaimida)

PubMed Central

Groza, Mariana; Lazarova, Stela; Luca, Francesca De; Fanelli, Elena; Milka Elshishka; Radoslavov, Georgi; Hristov, Peter; Coman, Mihaela; Peneva, Vlada

2017-01-01

Abstract Longidorus piceicola, a new geographical and host record from Romania, was described and illustrated on the basis of two populations originating from a coniferous and a deciduous forest. The main morphological characters of specimens from Romania correspond very well with the type material collected from the soil around Picea abies L. (Slovakia) except for the shorter body and tail. The D2-D3 fragment of 28S rDNA from both populations was amplified and sequenced, and the sequences were identical to L. piceicola sequence from Slovakia. The partial 18S-ITS1-5.8S-ITS2 rDNA regions from one of the populations were sequenced for the first time. The evolutionary relationships between L. piceicola and the closest species L. intermedius based on D2-D3 sequence divergence and single-nucleotide polymorphisms are discussed. Although having very low sequence dissimilarity (0.3–0.9 %) both species have distinct morphology and biology. Longidorus piceicola differs from L. intermedius in having a much longer odontostyle, body, distance anterior end - guide ring, a wider lip region, more ventromedian supplements (11 vs 5–7) in the male, and develops through four rather than three juvenile stages. Furthermore, L. piceicola occurs more frequently in association with conifers, while L. intermedius is found mainly in oak forests. PMID:28769632

PUTATIVE GENE PROMOTER SEQUENCES IN THE CHLORELLA VIRUSES

PubMed Central

Fitzgerald, Lisa A.; Boucher, Philip T.; Yanai-Balser, Giane; Suhre, Karsten; Graves, Michael V.; Van Etten, James L.

2008-01-01

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication. PMID:18768195

Isolation of an insertion sequence (IS1051) from Xanthomonas campestris pv. dieffenbachiae with potential use for strain identification and characterization.

PubMed Central

Berthier, Y; Thierry, D; Lemattre, M; Guesdon, J L

1994-01-01

A new insertion sequence was isolated from Xanthomonas campestris pv. dieffenbachiae. Sequence analysis showed that this element is 1,158 bp long and has 15-bp inverted repeat ends containing two mismatches. Comparison of this sequence with sequences in data bases revealed significant homology with Escherichia coli IS5. IS1051, which detected multiple restriction fragment length polymorphisms, was used as a probe to characterize strains from the pathovar dieffenbachiae. Images PMID:7906933

The complete chloroplast genome sequence of Euonymus japonicus (Celastraceae).

PubMed

Choi, Kyoung Su; Park, SeonJoo

2016-09-01

The complete chloroplast (cp) genome sequence of the Euonymus japonicus, the first sequenced of the genus Euonymus, was reported in this study. The total length was 157 637 bp, containing a pair of 26 678 bp inverted repeat region (IR), which were separated by small single copy (SSC) region and large single copy (LSC) region of 18 340 bp and 85 941 bp, respectively. This genome contains 107 unique genes, including 74 coding genes, four rRNA genes, and 29 tRNA genes. Seventeen genes contain intron of E. japonicus, of which three genes (clpP, ycf3, and rps12) include two introns. The maximum likelihood (ML) phylogenetic analysis revealed that E. japonicus was closely related to Manihot and Populus.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

PubMed

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-03-12

ZmbZIP25 ( Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter

PubMed Central

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-01-01

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from −2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from −2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5′-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5′-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5′-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5′-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from −1117 to −957 that were responsible for the specificity of the ZmbZIP25 5′-flanking sequence. PMID:29534529

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus Populus.

PubMed

Rajagopal, J; Das, S; Khurana, D K; Srivastava, P S; Lakshmikumaran, M

1999-10-01

This report aims to describe the identification and molecular characterization of a 145-bp tandem repeat family that accounts for nearly 1.5% of the Populus genome. Three members of this repeat family were cloned and sequenced from Populus deltoides and P. ciliata. The dimers of the repeat were sequenced in order to confirm the head-to-tail organization of the repeat. Hybridization-based analysis using the 145-bp tandem repeat as a probe on genomic DNA gave rise to ladder patterns which were identified to be a result of methylation and (or) sequence heterogeneity. Analysis of the methylation pattern of the repeat family using methylation-sensitive isoschizomers revealed variable methylation of the C residues and lack of methylation of the A residues. Sequence comparisons between the monomers revealed a high degree of sequence divergence that ranged between 6% and 11% in P. deltoides and between 4.2% and 8.3% in P. ciliata. This indicated the presence of sub-families within the 145-bp tandem family of repeats. Divergence was mainly due to the accumulation of point mutations and was concentrated in the central region of the repeat. The 145-bp tandem repeat family did not show significant homology to known tandem repeats from plants. A short stretch of 36 bp was found to show homology of 66.7% to a centromeric repeat from Chironomus plumosus. Dot-blot analysis and Southern hybridization data revealed the presence of the repeat family in 13 of the 14 Populus species examined. The absence of the 145-bp repeat from P. euphratica suggested that this species is relatively distant from other members of the genus, which correlates with taxonomic classifications. The widespread occurrence of the tandem family in the genus indicated that this family may be of ancient origin.

Physical mapping of repetitive DNA suggests 2n reduction in Amazon turtles Podocnemis (Testudines: Podocnemididae)

PubMed Central

Cavalcante, Manoella Gemaque; Bastos, Carlos Eduardo Matos Carvalho; Nagamachi, Cleusa Yoshiko; Pieczarka, Julio Cesar; Vicari, Marcelo Ricardo; Noronha, Renata Coelho Rodrigues

2018-01-01

Cytogenetic studies show that there is great karyotypic diversity in order Testudines (2n = 26–68), and that this may be mainly attributed to the presence/absence of microchromosomes. Members of the Podocnemididae family have the smallest diploid numbers of this order (2n = 26–28), which may be a derived condition of the group. Diverse studies suggest that repetitive-DNA-rich sites generally act as hotspots for double-strand breaks and chromosomal reorganization. In this context, we used fluorescent in situ hybridization (FISH) to map telomeric sequences (TTAGGG)n, 45S rDNA, and the genes encoding histones H1 and H3 in two species of genus Podocnemis. We also observed conservation of the 45S rDNA and H1 histone sequences (probable case of conserved synteny), but multiple conserved and non-conserved clusters of H3 genes, which colocalized with the interstitial telomeric sequences in the Podocnemis genome. Our results suggest that fusions have occurred between macro and microchromosomes or between microchromosomes, leading to the observed reduction in diploid number in the family Podocnemididae. PMID:29813087

Physical mapping of repetitive DNA suggests 2n reduction in Amazon turtles Podocnemis (Testudines: Podocnemididae).

PubMed

Cavalcante, Manoella Gemaque; Bastos, Carlos Eduardo Matos Carvalho; Nagamachi, Cleusa Yoshiko; Pieczarka, Julio Cesar; Vicari, Marcelo Ricardo; Noronha, Renata Coelho Rodrigues

2018-01-01

Cytogenetic studies show that there is great karyotypic diversity in order Testudines (2n = 26-68), and that this may be mainly attributed to the presence/absence of microchromosomes. Members of the Podocnemididae family have the smallest diploid numbers of this order (2n = 26-28), which may be a derived condition of the group. Diverse studies suggest that repetitive-DNA-rich sites generally act as hotspots for double-strand breaks and chromosomal reorganization. In this context, we used fluorescent in situ hybridization (FISH) to map telomeric sequences (TTAGGG)n, 45S rDNA, and the genes encoding histones H1 and H3 in two species of genus Podocnemis. We also observed conservation of the 45S rDNA and H1 histone sequences (probable case of conserved synteny), but multiple conserved and non-conserved clusters of H3 genes, which colocalized with the interstitial telomeric sequences in the Podocnemis genome. Our results suggest that fusions have occurred between macro and microchromosomes or between microchromosomes, leading to the observed reduction in diploid number in the family Podocnemididae.

Taxonomic Subgroups of Pasteurella multocida Correlate with Clinical Presentation

PubMed Central

Chen, Henry I.; Hulten, Kristina; Clarridge III, Jill E.

2002-01-01

Pasteurella multocida is a small nonmotile gram-negative coccobacillus that is found in the nasopharynx and gastrointestinal tract of many wild and domesticated animals. In humans it most commonly causes cellulitis and localized superficial skin abscesses following an animal bite or scratch. The respiratory tract is the second most common site of infection for Pasteurella. Of the more than 17 species of Pasteurella known, Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica are among the most common pathogens in humans. With the use of molecular techniques, distinction between different subspecies of P. multocida can be made more easily and accurately. We used the sequence of the 16S ribosomal DNA (rDNA) and repetitive extragenic palindromic sequence-PCR (REP-PCR) to characterize 20 strains (14 of P. multocida subsp. multocida and 6 of P. multocida subsp. septica; the 16S rDNA is identical for P. multocida subsp. multocida and Pasteurella multocida subsp. gallicida but differs from that of P. multocida subsp. septica) isolated from various anatomic sites. We found excellent correlation between the 16S rDNA sequence (a marker for a small conserved region of the genome), REP-PCR (a marker for a large portion of the genome), and biochemical tests (trehalose and sorbitol). We also found a correlation between the clinical presentation and the taxonomic group, with P. multocida subsp. septica more often associated with wounds than with respiratory infections (67 versus 17%, respectively) (P < 0.05, Z test) and P. multocida subsp. multocida more often associated with respiratory infections than with wounds (71 versus 14%, respectively) (P < 0.05, Z test). PMID:12202590

Taxonomy of oxalotrophic Methylobacterium strains

NASA Astrophysics Data System (ADS)

Sahin, Nurettin; Kato, Yuko; Yilmaz, Ferah

2008-10-01

Most of the oxalotrophic bacteria are facultative methylotrophs and play important ecological roles in soil fertility and cycling of elements. This study gives a detailed picture of the taxonomy and diversity of these bacteria and provides new information about the taxonomical variability within the genus Methylobacterium. Twelve mesophilic, pink-pigmented, and facultatively methylotrophic oxalate-oxidizing strains were included in this work that had been previously isolated from the soil and some plant tissues by the potassium oxalate enrichment method. The isolates were characterized using biochemical tests, cellular lipid profiles, spectral characteristics of carotenoid pigments, G+C content of the DNA, and 16S rDNA sequencing. The taxonomic similarities among the strains were analyzed using the simple matching ( S SM) and Jaccard ( S J) coefficients, and the UPGMA clustering algorithm. The phylogenetic position of the strains was inferred by the neighbor-joining method on the basis of the 16S rDNA sequences. All isolates were Gram-negative, facultatively methylotrophic, oxidase and catalase positive, and required no growth factors. Based on the results of numerical taxonomy, the strains formed four closely related clusters sharing ≥85% similarity. Analysis of the 16S rDNA sequences demonstrated that oxalotrophic, pink-pigmented, and facultatively methylotrophic strains could be identified as members of the genus Methylobacterium. Except for M. variabile and M. aquaticum, all of the Methylobacterium type strains tested had the ability of oxalate utilization. Our results indicate that the capability of oxalate utilization seems to be an uncommon trait and could be used as a valuable taxonomic criterion for differentiation of Methylobacterium species.

Taxonomy of oxalotrophic Methylobacterium strains.

PubMed

Sahin, Nurettin; Kato, Yuko; Yilmaz, Ferah

2008-10-01

Most of the oxalotrophic bacteria are facultative methylotrophs and play important ecological roles in soil fertility and cycling of elements. This study gives a detailed picture of the taxonomy and diversity of these bacteria and provides new information about the taxonomical variability within the genus Methylobacterium. Twelve mesophilic, pink-pigmented, and facultatively methylotrophic oxalate-oxidizing strains were included in this work that had been previously isolated from the soil and some plant tissues by the potassium oxalate enrichment method. The isolates were characterized using biochemical tests, cellular lipid profiles, spectral characteristics of carotenoid pigments, G+C content of the DNA, and 16S rDNA sequencing. The taxonomic similarities among the strains were analyzed using the simple matching (S (SM)) and Jaccard (S (J)) coefficients, and the UPGMA clustering algorithm. The phylogenetic position of the strains was inferred by the neighbor-joining method on the basis of the 16S rDNA sequences. All isolates were Gram-negative, facultatively methylotrophic, oxidase and catalase positive, and required no growth factors. Based on the results of numerical taxonomy, the strains formed four closely related clusters sharing > or =85% similarity. Analysis of the 16S rDNA sequences demonstrated that oxalotrophic, pink-pigmented, and facultatively methylotrophic strains could be identified as members of the genus Methylobacterium. Except for M. variabile and M. aquaticum, all of the Methylobacterium type strains tested had the ability of oxalate utilization. Our results indicate that the capability of oxalate utilization seems to be an uncommon trait and could be used as a valuable taxonomic criterion for differentiation of Methylobacterium species.

[Identification and phylogenetic application of unique nucleotide sequence of nad7 intron2 in Rhodiola (Crassulaceae) species].

PubMed

Deng, Ke-Jun; Yang, Zu-Jun; Liu, Cheng; Zhao, Wei; Liu, Chang; Feng, Juan; Ren, Zheng-Long

2007-03-01

Genetic characterization of 9 populations of Rhodiola crenulata, R. fastigiata and R. sachalinensis (Crassulaceae) species from Sichuan and Jilin Provinces of China, was investigated using the conserved primer of nad7 intron 2. All PCR products about 800 bp long were shorter than other Crassulaceae plants, which were used as molecular markers to identify the Rhodiola species. The sequence of the products indicated that total exon of 53 bp and intron of 738 bp exhibit only 9 nucleotide variations. Blasting the nad7 sequences to GenBank and the phylogenetic analysis showed that the sequence of Rhodiola species was clusted independently, and the length was smaller than all the registered sequences of higher plants. The result suggests that the Rhiodola species had a unique sequence in this gene region, which might be related to the special growth condition.

Primary analysis of repeat elements of the Asian seabass (Lates calcarifer) transcriptome and genome

PubMed Central

Kuznetsova, Inna S.; Thevasagayam, Natascha M.; Sridatta, Prakki S. R.; Komissarov, Aleksey S.; Saju, Jolly M.; Ngoh, Si Y.; Jiang, Junhui; Shen, Xueyan; Orbán, László

2014-01-01

As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8–14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates. PMID:25120555

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis.

PubMed

Masuda, Michiaki; Hirose, Noriyuki; Ishikawa, Tomohiro; Ikawa, Yoshiya; Nishimura, Kazuko

2016-03-01

Species of the genus Prototheca are achlorophyllous algae and ubiquitous in nature, and so far, six species have been listed in this genus: Prototheca wickerhamii, Prototheca zopfii, Prototheca blaschkeae, Prototheca cutis, Prototheca stagnora and Prototheca ulmea. A strain of the genus Prototheca, IFM 53848T, was isolated in Japan from a patient with systemic protothecosis and had been designated P. wickerhamii. Our previous study, by using PCR analysis, revealed that its SSU rRNA gene (rDNA) was distinctively larger than that of P. wickerhamii and other species of the genus Prototheca. In this study, molecular analysis showed that the exceptionally large SSU rDNA of IFM 53848T contains four group I introns. The morphology of IFM 53848T was indistinguishable from those of P. wickerhamii or P. cutis, and phylogenetic analyses, based on the sequences of the SSU rDNA exons and the D1/D2 region of the large subunit rDNA, indicated that IFM 53848T was closely related to P. cutis. On the other hand, unlike P. cutis, IFM 53848T failed to assimilate fructose or lysine and grew well at higher temperatures of up to 42 °C. In addition, the nucleotide sequence of the ribosomal internal transcribed spacer and the matrix assisted laser desorption ionization time-of-flight mass spectrometry profile of IFM 53848T were clearly distinct from those of P. cutis. The results strongly suggest that IFM 53848T represents a novel species, and so the seventh member of the genus Prototheca, which we have named Prototheca miyajii sp. nov. The unique characteristics of the strain may provide useful insights into the systematic taxonomy of the genus Prototheca.

Ribosomal Internal Transcribed Spacer of Prototheca wickerhamii Has Characteristic Structure Useful for Identification and Genotyping

PubMed Central

Hirose, Noriyuki; Nishimura, Kazuko; Inoue-Sakamoto, Maki; Masuda, Michiaki

2013-01-01

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms. PMID:24312279

Ribosomal internal transcribed spacer of Prototheca wickerhamii has characteristic structure useful for identification and genotyping.

PubMed

Hirose, Noriyuki; Nishimura, Kazuko; Inoue-Sakamoto, Maki; Masuda, Michiaki

2013-01-01

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms.

Functional analyses of PtRDM1 gene overexpression in poplars and evaluation of its effect on DNA methylation and response to salt stress.

PubMed

Movahedi, Ali; Zhang, Jiaxin; Sun, Weibo; Mohammadi, Kourosh; Almasi Zadeh Yaghuti, Amir; Wei, Hui; Wu, Xiaolong; Yin, Tongming; Zhuge, Qiang

2018-06-01

Epigenetic modification by DNA methylation is necessary for all cellular processes, including genetic expression events, DNA repair, genomic imprinting and regulation of tissue development. It occurs almost exclusively at the C5 position of symmetric CpG and asymmetric CpHpG and CpHpH sites in genomic DNA. The RNA-directed DNA methylation (RDM1) gene is crucial for heterochromatin and DNA methylation. We overexpressed PtRDM1 gene from Populus trichocarpa to amplify transcripts of orthologous RDM1 in 'Nanlin895' (P. deltoides × P. euramericana 'Nanlin895'). This overexpression resulted in increasing RDM1 transcript levels: by ∼150% at 0 mM NaCl treatment and by ∼300% at 60 mM NaCl treatment compared to WT (control) poplars. Genomic cytosine methylation was monitored within 5.8S rDNA and histone H3 loci by bisulfite sequencing. In total, transgenic poplars revealed more DNA methylation than WT plants. In our results, roots revealed more methylated CG contexts than stems and leaves whereas, histone H3 presented more DNA methylation than 5.8S rDNA in both WT and transgenic poplars. The NaCl stresses enhanced more DNA methylation in transgenic poplars than WT plants through histone H3 and 5.8 rDNA loci. Also, the overexpression of PtRDM1 resulted in hyper-methylation, which affected plant phenotype. Transgenic poplars revealed significantly more regeneration of roots than WT poplars via NaCl treatments. Our results proved that RDM1 protein enhanced the DNA methylation by chromatin remodeling (e.g. histone H3) more than repetitive DNA sequences (e.g. 5.8S rDNA). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

PubMed Central

Heilig, Hans G.H.J.; Zoetendal, Erwin G.; Vaughan, Elaine E.; Marteau, Philippe; Akkermans, Antoon D.L.; de Vos, Willem M.

2002-01-01

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract. PMID:11772617

The complete chloroplast genome sequence of Hibiscus syriacus.

PubMed

Kwon, Hae-Yun; Kim, Joon-Hyeok; Kim, Sea-Hyun; Park, Ji-Min; Lee, Hyoshin

2016-09-01

The complete chloroplast genome sequence of Hibiscus syriacus L. is presented in this study. The genome is composed of 161 019 bp in length, with a typical circular structure containing a pair of inverted repeats of 25 745 bp of length separated by a large single-copy region and a small single-copy region of 89 698 bp and 19 831 bp of length, respectively. The overall GC content is 36.8%. One hundred and fourteen genes were annotated, including 81 protein-coding genes, 4 ribosomal RNA genes and 29 transfer RNA genes.

Complete Genome Sequences of 38 Gordonia sp. Bacteriophages

PubMed Central

Montgomery, Matthew T.; Bonilla, J. Alfred; Dejong, Randall; Garlena, Rebecca A.; Guerrero Bustamante, Carlos; Klyczek, Karen K.; Russell, Daniel A.; Wertz, John T.; Jacobs-Sera, Deborah; Hatfull, Graham F.

2017-01-01

ABSTRACT We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity. PMID:28057748

The complete chloroplast genome of Capsicum annuum var. glabriusculum using Illumina sequencing.

PubMed

Raveendar, Sebastin; Na, Young-Wang; Lee, Jung-Ro; Shim, Donghwan; Ma, Kyung-Ho; Lee, Sok-Young; Chung, Jong-Wook

2015-07-20

Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

Identification and characterization of cell-specific enhancer elements for the mouse ETF/Tead2 gene.

PubMed

Tanoue, Y; Yasunami, M; Suzuki, K; Ohkubo, H

2001-12-21

We have identified and characterized by transient transfection assays the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF (Embryonic TEA domain-containing factor)/Tead2 gene required for transcriptional activation in ETF/Tead2 gene-expressing cells, such as P19 cells. The 117-bp enhancer contains one GC-rich sequence (5'-GGGGCGGGG-3'), termed the GC box, and two tandemly repeated GA-rich sequences (5'-GGGGGAGGGG-3'), termed the proximal and distal GA elements. Further analyses, including transfection studies and electrophoretic mobility shift assays using a series of deletion and mutation constructs, indicated that Sp1, a putative activator, may be required to predominate over its competition with another unknown putative repressor, termed the GA element-binding factor, for binding to both the GC box, which overlapped with the proximal GA element, and the distal GA element in the 117-bp sequence in order to achieve a full enhancer activity. We also discuss a possible mechanism underlying the cell-specific enhancer activity of the 117-bp sequence.

Morphological and Phylogenetic Characterization of New Gephyrocapsa Isolates Suggests Introgressive Hybridization in the Emiliania/Gephyrocapsa Complex (Haptophyta).

PubMed

Bendif, El Mahdi; Probert, Ian; Young, Jeremy R; von Dassow, Peter

2015-07-01

The coccolithophore genus Gephyrocapsa contains a cosmopolitan assemblage of pelagic species, including the bloom-forming Gephyrocapsa oceanica, and is closely related to the emblematic coccolithophore Emiliania huxleyi within the Noëlaerhabdaceae. These two species have been extensively studied and are well represented in culture collections, whereas cultures of other species of this family are lacking. We report on three new strains of Gephyrocapsa isolated into culture from samples from the Chilean coastal upwelling zone using a novel flow cytometric single-cell sorting technique. The strains were characterized by morphological analysis using scanning electron microscopy and phylogenetic analysis of 6 genes (nuclear 18S and 28S rDNA, plastidial 16S and tufA, and mitochondrial cox1 and cox3 genes). Morphometric features of the coccoliths indicate that these isolates are distinct from G. oceanica and best correspond to G. muellerae. Surprisingly, both plastidial and mitochondrial gene phylogenies placed these strains within the E. huxleyi clade and well separated from G. oceanica isolates, making Emiliania appear polyphyletic. The only nuclear sequence difference, 1bp in the 28S rDNA region, also grouped E. huxleyi with the new Gephyrocapsa isolates and apart from G. oceanica. Specifically, the G. muellerae morphotype strains clustered with the mitochondrial β clade of E. huxleyi, which, like G. muellerae, has been associated with cold (temperate and sub-polar) waters. Among putative evolutionary scenarios that could explain these results we discuss the possibility that E. huxleyi is not a valid taxonomic unit, or, alternatively the possibility of past hybridization and introgression between each E. huxleyi clade and older Gephyrocapsa clades. In either case, the results support the transfer of Emiliania to Gephyrocapsa. These results have important implications for relating morphological species concepts to ecological and evolutionary units of diversity. Copyright © 2015 Elsevier GmbH. All rights reserved.

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

NASA Astrophysics Data System (ADS)

Zhao, Cui; Zhang, Xiaojun; Liu, Chengzhang; Huan, Pin; Li, Fuhua; Xiang, Jianhai; Huang, Chao

2012-05-01

Little is known about the genome of Pacific white shrimp ( Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 pairedends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

PubMed Central

Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi

2014-01-01

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. PMID:24344172

Biodiversity and molecular ecology of Russula and Lactarius in Alaska based on soil and sporocarp DNA sequences

Treesearch

Geml J.; D.L. Taylor

2013-01-01

Although critical for the functioning of ecosystems, fungi are poorly known in highlatitude regions. This paper summarizes the results of the first genetic diversity assessments of Russula and Lactarius, two of the most diverse and abundant fungal genera in Alaska. SU rDNA sequences from both curated sporocarp collections and soil PCR clone libraries sampled in...

Positive Streptobacillus moniliformis PCR in guinea pigs likely due to Leptotrichia spp.

PubMed

Boot, Ron; Van de Berg, Lia; Reubsaet, Frans A G; Vlemminx, Maurice J

2008-04-30

Streptobacillus moniliformis is a zoonotic bacterium. We obtained positive S. moniliformis PCR results in oral swab samples from guinea pigs from an experimental colony and the breeding colony of origin. Comparison of the DNA sequence of an amplicon with deposited 16S rDNA sequences revealed that Leptotrichia sp. can be the source of a false positive S. moniliformis PCR outcome.

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae.

PubMed

Sloan, Roketa; Huang, Shar-Yin Naomi; Pommier, Yves; Jinks-Robertson, Sue

2017-11-01

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasmid origin of replication of herpesvirus papio: DNA sequence and enhancer function.

PubMed Central

Loeb, D D; Sung, N S; Pesano, R L; Sexton, C J; Hutchison, C; Pagano, J S

1990-01-01

Herpesvirus papio (HVP) is a lymphotropic virus of baboons which is related to Epstein-Barr virus (EBV) and produces latent infection. The nucleotide sequence of the 5,775-base-pair (bp) EcoRI K fragment of HVP, which has previously been shown to confer the ability to replicate autonomously, has been determined. Within this DNA fragment is a region which bears structural and sequence similarity to the ori-P region of EBV. The HVP ori-P region has a 10- by 26-bp tandem array which is related to the 20- by 30-bp tandem array from the EBV ori-P region. In HVP there is an intervening region of 764 bp followed by five partial copies of the 26-bp monomer. Both the EBV and HVP 3' regions have the potential to form dyad structures which, however, differ in arrangement. We also demonstrate that a transcriptional enhancer which requires transactivation by a virus-encoded factor is present in the HVP ori-P. Images PMID:2159548

Assessing Subsurface Bioaugmentation of a Mixed Culture Capable of Chlorinated Solvent Cometabolism via Molecular Methods

NASA Astrophysics Data System (ADS)

Dolan, M. E.; Lim, H. K.; Semprini, L.; Giovanonni, S.; Vergin, K.; McCarty, P. L.; Hopkins, G. D.

2001-12-01

The goal of this project is the successful bioaugmentation of a mixed culture capable of aerobic cometabolism of chlorinated solvent mixtures into an aquifer test zone at Moffett Federal Airfield, CA (Moffett). The test zone consists of two parallel well legs both fed butane and oxygen. One leg will be bioaugmented and the other will serve as an indigenous control. Injection and extraction wells and six (3 per leg) intermediately placed groundwater monitoring points will be frequently monitored for chlorinated solvents, butane, dissolved oxygen, and pH. Groundwater will also be periodically analyzed for microbial content using terminal restriction fragment length polymorphism (T-RFLP) and fluorescence in-situ hybridization (FISH) analyses. In each well leg, two fully-penetrating wells containing solid media will be periodically analyzed for microbial colonization (T-RFLP). The mixed bioaugmentation culture originated from environmental samples taken from Hanford, WA. The culture was enriched on butane and tested for viability in Moffett groundwater and aquifer solids. A clone library was created from the 16S rDNA in the mixed culture and 86 clones were sorted based on RFLP patterns. Complete sequencing of the 16S rDNA gene from the three most prevalent clones revealed 45 clones similar to Acidovorax or Hydrogenophaga, gram negative proteobacterium, and 12 clones similar to Rhodococcus, a gram positive filamentous organism. Fluorescently-labeled rRNA probes were designed for FISH analyses and appropriate restriction enzymes were chosen for T-RFLP analyses based upon the sequence information. Microcosm tests were conducted prior to the initiation of the field study to evaluate butane, 1,1-dichloroethylene (1,1-DCE), and 1,1,1-trichloroethane (TCA) degradation kinetics and microbial community composition. Bioaugmented microcosms began butane utilization sooner than non-bioaugmented ones in the presence and absence of 1,1-DCE, and were able to degrade more 1,1-DCE (up to 500 Yg/L) faster than non-bioaugmented microcosms. T-RFLP analyses of triplicate bottles produced very consistent results. An organism(s) with a T-RFLP signature of 183 bp was found to dominate in bioaugmented microcosms and was consistently absent from non-bioaugmented microcosms. T-RFLP and FISH analyses of groundwater and solid media during the bioaugmentation field demonstration are expected to reveal the extent of transport and subsurface colonization of the bioaugmentation culture.

Divergent homologs of the predicted small RNA BpCand697 in Burkholderia spp.

NASA Astrophysics Data System (ADS)

Damiri, Nadzirah; Mohd-Padil, Hirzahida; Firdaus-Raih, Mohd

2015-09-01

The small RNA (sRNA) gene candidate, BpCand697 was previously reported to be unique to Burkholderia spp. and is encoded at 3' non-coding region of a putative AraC family transcription regulator gene. This study demonstrates the conservation of BpCand697 sequence across 32 Burkholderia spp. including B. pseudomallei, B. mallei, B. thailandensis and Burkholderia sp. by integrating both sequence homology and secondary structural analyses of BpCand697 within the dataset. The divergent sequence of BpCand697 was also used as a discriminatory power in clustering the dataset according to the potential virulence of Burkholderia spp., showing that B. thailandensis was clearly secluded from the virulent cluster of B. pseudomallei and B. mallei. Finally, the differential co-transcript expression of BpCand697 and its flanking gene, bpsl2391 was detected in Burkholderia pseudomallei D286 after grown under two different culture conditions using nutrient-rich and minimal media. It is hypothesized that the differential expression of BpCand697-bpsl2391 co-transcript between the two standard prepared media might correlate with nutrient availability in the culture media, suggesting that the physical co-localization of BpCand697 in B. pseudomallei D286 might be directly or indirectly involved with the transcript regulation of bpsl2391 under the selected in vitro culture conditions.

The complete plastid genome sequence of Eustrephus latifolius (Asparagaceae: Lomandroideae).

PubMed

Kim, Hyoung Tae; Kim, Jung Sung; Kim, Joo-Hwan

2016-01-01

The complete chloroplast (cp) genome sequence of Eustrephus latifolius was firstly determined in subfamily Lomandriodeae of family Asparagaceae. It was 159,736 bp and contained a large single copy region (82,403 bp) and a small single copy region (13,607 bp) which were separated by two inverted repeat regions (31,863 bp). In total, 132 genes were identified and they were consisted of 83 coding genes, 8 rRNA genes, 38 tRNA genes, 3 pseudogenes. rpl23 and clpP were pseudogenes due to sequence deletions. Among 23 genes containing introns, rps12 and ycf3 contained two introns and the rest had just one intron. The intact ycf68 was identified within an intron of trnI-GAU. The amino acid sequence was almost identical with Phoenix dactylifera in Aracales. Ycf1 of E. latifolius was completely located in IR. It was similar to cp genome structure of Lemna minor, Spirodela polyrhiza, Wolffiella lingulata, Wolffia australiana in Alismatales.

Determination of Trichuris skrjabini by sequencing of the ITS1-5.8S-ITS2 segment of the ribosomal DNA: comparative molecular study of different species of trichurids.

PubMed

Cutillas, C; Oliveros, R; de Rojas, M; Guevara, D C

2004-06-01

Adults of Trichuris skrjahini have been isolated from the cecum of caprine hosts (Capra hircus), Trichuris ovis and Trichuris globulosa from Ovis aries (sheep) and C. hircus (goats), and Trichuris leporis from Lepus europaeus (rabbits) in Spain. Genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced by polymerase chain reaction (PCR) techniques. The ITS1 of T. skrjabini, T. ovis, T. globulosa, and T. leporis was 495, 757, 757, and 536 nucleotides in length, respectively, and had G + C contents of 59.6, 58.7, 58.7, and 60.8%, respectively. Intraindividual variation was detected in the ITSI sequences of the 4 species. Furthermore, the 5.8S sequences of T. skrjabini, T. ovis, T. globulosa, and T. leporis were compared. A total of 157, 152, 153, and 157 nucleotides in length was observed in the 5.8S sequences of these 4 species, respectively. There were no sequence differences of ITS1 and 5.8S products between T. ovis and T. globulosa. Nevertheless, clear differences were detected between the ITS1 sequences of T. skrjabini, T. ovis, T. leporis, Trichuris muris, and T. arvicolae. The ITS2 fragment from the rDNA of T. skrjabini was sequenced. A comparative study of the ITS2 sequence of T. skrjabini with the previously published ITS2 sequence data of T. ovis, T. leporis, T. muris, and T. arvicolae suggested that the combined use of sequence data from both spacers would be useful in the molecular characterization of trichurid parasites.

Bioinformatics analysis and detection of gelatinase encoded gene in Lysinibacillussphaericus

NASA Astrophysics Data System (ADS)

Repin, Rul Aisyah Mat; Mutalib, Sahilah Abdul; Shahimi, Safiyyah; Khalid, Rozida Mohd.; Ayob, Mohd. Khan; Bakar, Mohd. Faizal Abu; Isa, Mohd Noor Mat

2016-11-01

In this study, we performed bioinformatics analysis toward genome sequence of Lysinibacillussphaericus (L. sphaericus) to determine gene encoded for gelatinase. L. sphaericus was isolated from soil and gelatinase species-specific bacterium to porcine and bovine gelatin. This bacterium offers the possibility of enzymes production which is specific to both species of meat, respectively. The main focus of this research is to identify the gelatinase encoded gene within the bacteria of L. Sphaericus using bioinformatics analysis of partially sequence genome. From the research study, three candidate gene were identified which was, gelatinase candidate gene 1 (P1), NODE_71_length_93919_cov_158.931839_21 which containing 1563 base pair (bp) in size with 520 amino acids sequence; Secondly, gelatinase candidate gene 2 (P2), NODE_23_length_52851_cov_190.061386_17 which containing 1776 bp in size with 591 amino acids sequence; and Thirdly, gelatinase candidate gene 3 (P3), NODE_106_length_32943_cov_169.147919_8 containing 1701 bp in size with 566 amino acids sequence. Three pairs of oligonucleotide primers were designed and namely as, F1, R1, F2, R2, F3 and R3 were targeted short sequences of cDNA by PCR. The amplicons were reliably results in 1563 bp in size for candidate gene P1 and 1701 bp in size for candidate gene P3. Therefore, the results of bioinformatics analysis of L. Sphaericus resulting in gene encoded gelatinase were identified.

Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients.

PubMed

Banko, Ana; Lazarevic, Ivana; Cupic, Maja; Stevanovic, Goran; Boricic, Ivan; Jovanovic, Tanja

2012-04-01

Seven strains of Epstein-Barr virus (EBV) are defined based on C-terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30-bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C-terminal region of LMP1 in Serbian isolates. This study included 53 EBV-DNA-positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506-bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non-deleted (66%), and the rest had 30-bp, rare 69-bp, or yet unknown 27-bp deletions, which were not related to malignant or non-malignant isolate origin. However, the majority of 69-bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33-bp repeats were found in the majority of non-deleted isolates (68.6%), whereas most 69-bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95-8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical-associated characteristics in the LMP1 C terminus of investigated isolates. Copyright © 2012 Wiley Periodicals, Inc.

Molecular marker to identify radiolarian species -toward establishment of paleo-environmental proxy-

NASA Astrophysics Data System (ADS)

Ishitani, Y.

2017-12-01

Marine fossilized unicellular plankton are known to have many genetically divergent species (biological species) in the single morphological species and these biological species show the species-specific environments much more precisely than that of morphological species. Among these plankton, Radiolaria are one of the best candidates for time- and environmental-indicators in the modern and past oceans, because radiolarians are the only group which represent entire water column from shallow to deep waters. However, the ecology and evolution of radiolarian were traditionally studied in paleontology and paleoceanography by morphological species. Even Radiolaria has a huge potential for novel proxy of wide and deep environments, there is no criterion to identify the biological species. The motivation for this study is setting the quantitative delimitation to establish the biological species of radiolarians based on molecular data, for leading the future ecological and paleo-environmental study. Identification of the biological species by ribosomal DNA sequences are mainly based on two ways: one is the evolutionary distance of the small subunit (SSU) rDNA, the internal transcribed spacer region of ribosomal DNA (ITS1 and 2), and the large subunit (LSU) rDNA; and the other is the secondary structure of ITS2. In the present study, all four possible genetic markers (SSU, ITS1, ITS2, and LSU rDNA) were amplified from 232 individuals of five radiolarian morphological species and applied to examine the evolutionary distance and secondary structure of rDNA. Comprehensive survey clearly shows that evolutionary distance of ITS1 rDNA and the secondary structure of ITS2 is good to identify the species. Notably, evolutionary distance of ITS1 rDNA is possible to set the common delimitation to identify the biological species, as 0.225 substitution per site. The results show that the ITS1 and ITS 2 rDNA could be the criterion for radiolarian species identification.