rDNA Intergenic Spacer Research Articles

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum β-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

The Population of Fusarium oxysporum f. sp. lactucae in California and Arizona.

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae, is now found in all major lettuce producing regions in California and Arizona. The population structure of F. oxysporum f. sp. lactucae in California and Arizona was characterized based on somatic compatibility and sequences of the translation elongation factor 1-α gene (EF-1α) and rDNA intergenic spacer region (IGS). In this study, 170 isolates were tested for somatic compatibility based on heterokaryon formation, using complementary nitrate nonutilizing (nit) mutants. Five subgroups (A to E) of somatic compatibility group 0300 were identified. Isolates associated with the same subgroup had a strong complementation reaction, whereas reactions between isolates of different subgroups were weak or delayed. An isolate from the first known infestation of Fusarium wilt of lettuce in California was associated with subgroup A, which predominated among isolates in our collection. Isolates representative of each subgroup were confirmed to be associated with race 1, based on the reaction of differential lettuce cultivars. It is possible that somatic compatibility subgroups B to E of F. oxysporum f. sp. lactucae were derived from subgroup A, as a consequence of somatic mutations affecting compatibility. If so, subgroups of F. oxysporum f. sp. lactucae may represent an intermediate step in divergence that will lead to clearly separable compatibility groups. Sequences of EF-1α and IGS were both identical for 58 isolates of F. oxysporum f. sp. lactucae that represented all somatic compatibility subgroups and locations from which isolates were obtained, indicating that subgroups were derived from the same clonal lineage (VCG 0300).

Intergenic Spacer Single Nucleotide Polymorphisms for Genotyping Amylostereum areolatum (Russulales: Amylostereacea) Symbionts of Native and Non-native Sirex Species

Abstract In North America Amylostereum areolatum (Chaillet ex Fr.) Boidin is a fungal symbiont associated with both the non-native Sirex noctilio Fabricius (Hymenoptera: Siricidae) and less commonly the native Sirex nigricornis Fabricius (Hymenoptera: Siricidae) woodwasps. The relationship between S. noctilio and A. areolatum constitutes a serious threat to pine plantation in the southern hemisphere. Studies have shown evidence of exchange of symbionts between non-native and native Sirex species. Our objectives were 1) to identify and assemble a panel of rDNA intergenic spacer–single nucleotide polymorphisms (IGS-SNPs) for genotyping strains of A. areolatum symbionts associated with Sirex species in North America, and 2) to develop genetic markers for monitoring the spread of specific A. areolatum haplotypes associated with S. noctilio across regions. The IGS-SNPs panel analyzed included haplotypes B1, B2, D1, D2 (from known IGS type B and D), E, and F. Genetic markers and haplotype-specific primers were designed to detect the IGS haplotypes D and E of A. areolatum. We found that haplotype D was absent in A. areolatum from S. nigricornis in Louisiana, while haplotype E was detected in all A. areolatum from S. nigricornis in Canada and Louisiana. Both haplotype D and E were co-detected in approximately 5% of samples from Canada. The IGS-SNP markers detected specific haplotypes accurately. Observing haplotype D in any A. areolatum from the native S. nigricornis likely indicates the presence of the potentially harmful S. noctilo-A. areolatum complex. The work highlights how IGS-SNPs can help in early detection without direct occurrence/observations of the non-native species of concern.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S–23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated.The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6.Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers’ health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Strong genetic structure in Dyckia excelsa (Bromeliaceae), an endangered species found on ironstone outcrops in Pantanal, Brazil

AbstractThe plant communities associated with iron-rich outcrops are still little known about to their genetic structure and diversity. Outcrops are often considered to be ‘terrestrial islands’, and gene flow among populations on different outcrops is presumed to be hampered by the isolation effect provided by the surrounding matrix. Here, we studied the genetic diversity and structure of populations of Dyckia excelsa to test the hypothesis that the disjunction of the ironstone outcrops where this species occurs promotes its highly structured genetic variability. Seven nuclear microsatellite markers and two plastid DNA intergenic spacers (rps16-trnK and rpl32-trnL) were used to characterize individuals from seven locations in the region of Urucum Residual Plateau, Mato Grosso do Sul State, Brazil. These markers low haplotype and allelic diversity and high levels of genetic divergence among outcrops, indicating prolonged genetic isolation, with diversification of haplotypes dating from the Pleistocene (1.0 Mya). Pollen movement may occur between neighbouring populations, resulting in a pattern of isolation-by-distance. For conservation purposes, it is recommended that D. excelsa be preserved in situ in each specific location given the low levels of gene flow and the high degree of genetic uniqueness in each group of populations investigated.

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow‐pigmented bacteria isolated from blight‐affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re‐emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.

BIOLOGICAL SOLUBILIZATION OF SOME METALS BY A NEW ACIDITHIOBACILLUS SPECIES ISOLATED FROM A MODERATE SULFUR HOT SPRING

A chemolithotrophic bacterium was isolated from sulfur hot spring. According to phenotypic traits and 16-23S rDNA intergenic spacer region analysis, the isolate was identified and named as Acidithiobacillus sp. MR39, which was a gram-negative, rod- shape and non-motile bacterium. The strain was able to grow in a synthetic liquid medium supplemented with the mineral ore as the source of energy. The optimum conditions were found to be within initial pH range of 2.0-2.5, at 34±1˚C and with shaking at 120 rpm. The bacterium had a remarkable potential for mineralization of 88% iron, 75% copper, 59% zinc, 59% nickel and 40% cobalt upon their growth in the liquid media. After adapting the bacterial cells to copper ions in 100 mM for 5-day incubation, biorecovery of Cu increased about 10% comparing to unadapted cells that are able to dissolve approximately 15% of total cu of mineral concentrate. Considering the finding in this study, the strain MR39 offers a great prospect for in situ extraction of metals from various ores along with other indigenous bacteria that can grow under ambient conditions.

5S Ribosomal DNA of Distantly Related Quercus Species: Molecular Organization and Taxonomic Application

The genus Quercus (oak) is a widespread and economically important genus of tree plants. The intrageneric taxonomy of this group remains controversial due to widely distributed interspecific hybridization and convergent similarity of morphological traits. Further progress in this problem requires application of molecular methods. Taking into account that the comparison of 5S rDNA was successfully used in the molecular taxonomy of plants, we cloned and sequenced this genomic region for representatives of three taxo-nomically distant Quercus species: Q. acutissima (sect. Cerris) and Q. glauca (sect. Cyclobalanopsis) from East Asia and Q. texana (sect. Lobatae) from North America. We also identified 5S rDNA in the genome sequences available in the Genbank database for North American species Q. lobata (sect. Quercus) and for the related genus Castanea. It was found that the 5S rDNA repeated units demonstrate a high level of intragenomic sequence similarity in representatives of genus Quercus. The potential external elements of the 5S rDNA promoter differ from those in other families of dicotyledonous plants. The results of the comparative sequence analysis of the 5S rDNA intergenic spacer region support the existing taxonomy of the genus and indicate the isolated position of section Cyclobalanopsis, which can be considered as a separate subgenus.

Subrepeats in 5s rDNAs as a molecular marker in Acer platanoides L. populations

Aim. To study the genetic diversity of tree species it is necessary to use only those regions of genome, which evolve at the highest rate, such as 5S rDNA. To estimate the potential of 5S rDNA to be used as a molecular marker for genogeographic studies, the molecular organization of this genomic region was compared between samples from two geogra-phically remote Bulgarian and Ukrainian populations of Norway maple, Acer platanoides. Methods. PCR amplification, cloning and sequencing. Results. It was shown that in the genome of A. platanoides the 5S rDNA sequences are highly similar. However, in the 5S rDNA intergenic spacer (IGS) of A. platanoides from the Bulgarian population three copies of GTCCGTT subrepeats are present, whereas only one copy of this sequence occurs in plants from the Ukrainian population. Except for different number of subrepeats, the 5S rDNA sequences of the Bulgarian and Ukrainian samples of A. platanoides are identical. In two taxonomically distant species, A. platanoides and A. pseudoplatanus, the region of 5S rDNA IGS, which contains the potential external elements of the promoter of RNA polymerase III, shows no difference, and therefore evolves at a lower rate than other parts of the IGS. Conclusions. The identity of the potential external promoter elements within the genus Acer supports the idea that this part of the IGS could be involved in the initiation of 5S rDNA transcription. The presence of different numbers of sub-repeats in the 5S rDNA IGS in various populations of A. platanoides makes them suitable for identification of intraspecific forms and for evaluation of the intraspecific genetic diversity of A. platanoides.
 Keywords: 5S rDNA, molecular evolution, sub-repetitions, intraspecific variability, Acer.

Intraspecific genetic variation in Trichinella spiralis and Trichinella britovi populations circulating in different geographical regions of Poland.

Trichinella spiralis and Trichinella britovi are species of nematodes which are responsible for the majority of Trichinella infections in the world and the most prevalent in Poland. The most abundant species – T. spiralis, is considered to be more genetically homogeneous in Europe than T. britovi. The aim of the present study was to determine the genetic variability in T. spiralis and T. britovi populations based on nuclear 5S rDNA intergenic spacer region (5S rDNA) and cytochrome c oxidase 1 (COX1) gene sequences. For the study, 55 isolates of T. spiralis and 50 isolates of T. britovi isolated from wild boars, pigs, brown rat and a red fox were analyzed. Based on the analysis of both genes, the genetic variability within populations of T. spiralis and T. britovi differed. In T. spiralis, two single nucleotide polymorphisms (SNPs) were observed in the 612 bp 5S rDNA gene fragment, and one SNP was detected in the 700 bp COX1 gene fragment. In T. britovi, 17 single nucleotide variations (SNVs) were detected in the 5S rDNA gene fragment (among them 16 SNPs), while COX1 sequence analysis revealed the occurrence of 20 SNVs between the sequences tested (among them 19 SNPs).For the majority of T. spiralis isolates the investigated larvae presented uniform haplotypes. In contrast, most of the isolates of T. britovi consisted of larvae of different haplotypes. Geographical analysis showed that each region exhibited different haplotype composition and richness. Warmińsko-Mazurskie and Zachodniopomorskie regions were the richest in haplotypes (15 and 16 haplotypes, respectively). We used heatmaps showing a characteristic pattern for each region graphically. This may allow to differentiate regions based on the occurrence of particular haplotypes. Furthermore, a PCA analysis on the SNP level yielded biplots that show that certain haplotypes/genotypes are associated with (clusters of) regions.

Life time of some RNA products of rDNA intergenic spacer in HeLa cells.

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

Life time of some products of human rDNA intergenic spacer

Life time of some products of human rDNA intergenic spacer

Soybean Sudden Death Syndrome Causal Agent Fusarium brasiliense Present in Michigan.

Sudden death syndrome (SDS), caused by members of Fusarium solani species complex (FSSC) clade 2, is a major and economically important disease in soybean worldwide. The primary causal agent of SDS isolated to date in North America has been F. virguliforme. In 2014 and 2016, SDS symptoms were found in two soybean fields located on the same farm in Michigan. Seventy Fusarium strains were isolated from roots of the SDS-symptomatic soybeans in two fields. Phylogenetic analysis of partial sequences of elongation factor-1α, the nuclear ribosomal DNA intergenic spacer region, and the RNA polymerase II beta subunit revealed that the primary FSSC species isolated was F. brasiliense (58 and 36% in each field) and the remaining Fusarium strains were identified as F. cuneirostrum, F. phaseoli, an undescribed Fusarium sp. from FSSC clade 2, and strains in FSSC clade 5 and FSSC clade 11. Molecular identification was supported with morphological analysis and a pathogenicity assay. The soybean seedling pathogenicity assay indicated that F. brasiliense was capable of causing typical foliar SDS symptoms. Both root rot and foliar disease severity were variable by strain, just as they are in F. virguliforme. Both FSSC 5 and FSSC 11 strains were also capable of causing root rot, but SDS foliar symptoms were not detected. To our knowledge, this is the first report of F. brasiliense causing SDS in soybean in the United States and the first report of F. cuneirostrum, F. phaseoli, an as-yet-unnamed Fusarium sp., and strains in FSSC clade 5 and FSSC clade 11 associated with or causing root rot of soybean in Michigan.

Genospecies of Borrelia burgdorferi sensu lato detected in 16 mammal species and questing ticks from northern Europe

Lyme borreliosis is the most common vector-borne zoonosis in the northern hemisphere, and the pathogens causing Lyme borreliosis have distinct, incompletely described transmission cycles involving multiple host groups. The mammal community in Fennoscandia differs from continental Europe, and we have limited data on potential competent and incompetent hosts of the different genospecies of Borrelia burgdorferi sensu lato (sl) at the northern distribution ranges where Lyme borreliosis is emerging. We used qPCR to determine presence of B. burgdorferi sl in tissue samples (ear) from 16 mammalian species and questing ticks from Norway, and we sequenced the 5S–23 S rDNA intergenic spacer region to determine genospecies from 1449 qPCR-positive isolates obtaining 423 sequences. All infections coming from small rodents and shrews were linked to the genospecies B. afzelii, while B. burgdorferi sensu stricto (ss) was only found in red squirrels (Sciurus vulgaris). Red squirrels were also infected with B. afzelii and B. garinii. There was no evidence of B. burgdorferi sl infection in moose (Alces alces), red deer (Cervus elaphus) or roe deer (Capreolus capreolus), confirming the role of cervids as incompetent hosts. In infected questing ticks in the two western counties, B. afzelii (67% and 75%) dominated over B. garinii (27% and 21%) and with only a few recorded B. burgdorferi ss and B. valaisiana. B. burgdorferi ss were more common in adult ticks than in nymphs, consistent with a reservoir in squirrels. Our study identifies potential competent hosts for the different genospecies, which is key to understand transmission cycles at high latitudes of Europe.

Patterns of genetic variation reflect multiple introductions and pre-admixture sources of common ragweed (Ambrosia artemisiifolia) in China

Ambrosia artemisiifolia is native to North America but has become a worldwide invasive weed. It was introduced to China more than 80 years ago and has spread into 20 provinces since then. To assess the population structure of A. artemisiifolia in China and whether this invasion involved a single event or multiple events, we investigated patterns of genetic variation for three chloroplast DNA intergenic spacer regions, a nrITS region and five microsatellite loci. Our dataset consists of 370 individuals from 19 sites throughout China. We compared their cpDNA-haplotypes to those published for native North American populations. The distribution of cpDNA-haplotypes indicates that A. artemisiifolia was introduced to China multiple times from different source regions. The numbers of alleles in Chinese populations were not significantly lower than in native populations. Both nrITS-haplotypes and microsatellite alleles showed that there was no evidence for a genetic bottleneck. Four populations were genetically well separated from the other 15 populations. However, the absence of isolation by distance, and the low levels of genetic differentiation and gene flow among the other 15 population suggest that most populations in China come from pre-admixed populations. To find the exact source regions of the Chinese populations, more samples from the native region and other invaded regions will be necessary. Nevertheless, our study provides important insights into the genetic background of A. artemisiifolia invasion in China.

Characterization analysis of the 35S rDNA intergenic spacers in Erianthus arundinaceus

The 35S ribosomal DNA (rDNA) units that occur in tandem repeat are separated by an intergenic spacer (IGS) that plays an important role in rRNA transcription. Moreover, IGS is an important molecular marker for evolutionary research in plants. In the present study, the IGS sequence of Erianthus arundinaceus was isolated and sequenced for the first time. Structure analysis indicated the entire IGS sequence of three typical E. arundinaceus genotypes was highly conserved, with approximately 3087 bp and 67.1% mean GC content. The putative transcription termination, and initiation sites as well as a large number of methylation sites were found to be present in the IGS of E. arundinaceus compared to other plants. The phylogenic tree constructed using the E. arundinaceus IGS sequence showed that Miscanthus sinensis var. glaber was genetically close to Saccharum spp. while E. arundinaceus was close to Imperata cylindrica. Moreover, fluorescent in situ hybridization revealed that IGS and pTa71 probes had the same locus at nucleolar organizer regions. Taken together, this work enhances our current understanding of the organization of IGS in E. arundinaceus and provides a molecular evidence for an evolutionary relationship between Saccharum spp., E. arundinaceus, I. cylindrica and M. sinensis var. glaber.

Intraspecific divergences and phylogeography of Panzerina lanata (Lamiaceae) in northwest China.

Climatic fluctuations during the Quaternary significantly affect many species in their intraspecific divergence and population structure across northwest China. In order to investigate the impact of climate change on herbaceous plants, we studied Panzerina lanata (Lamiaceae), a widely distributed species. Sequences of two chloroplast DNA (cpDNA) intergenic spacers (trnH-psbA and rpoB-trnC) and a nuclear ribosomal region (nrDNA, ITS) were generated from 27 populations of Panzerina lanata and resulted in the identification of seven chloroplast haplotypes and thirty-two nuclear haplotypes. We applied AMOVA, neutrality test and mismatch distribution analysis to estimate genetic differentiation and demographic characteristics. The divergence times of the seven cpDNA haplotypes were estimated using BEAST. Our results revealed high levels of genetic diversity (cpDNA: Hcp = 0.6691, HT = 0.673; nrDNA: Hnr = 0.5668, HT = 0.577). High level of genetic differentiation (GST = 0.950) among populations was observed in the cpDNA sequences, while the genetic differentiation values (GST = 0.348) were low in nuclear sequences. AMOVA results revealed major genetic variation among the three groups: northern, central, and eastern group. However, the genetic differentiation in ITS data was not found. The species distribution modeling and demographic analysis indicated that P. lanata had not experienced recent range expansion. The occurrence of divergence between seven cpDNA haplotypes, probably during Pleistocene, coincides with aridification and expansion of the desert across northwest China that resulted in species diversification and habitat fragmentation. In addition, we discovered that the deserts and the Helan Mountains acted as effective geographic barriers that promoting the intraspecific diversity of P. lanata.

Characterization and Differentiation of Chicken Mycoplasma Isolates Using 16S-23S Intergenic Spacer Region Sequencing

ABSTRACT The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: Sao Paulo, Minas Gerais, Rio de Janeiro and Espirito Santo. The isolates were recovered from the Sao Paulo, Rio de Janeiro and Espirito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

Application of random amplified polymorphism DNA and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism to predict major Streptococcus suis clonal complexes isolated from humans and pigs

Random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied and evaluated to determine clonal complexes (CCs) of 684 Streptococcus suis isolates from pigs and humans. RAPD better distinguished major S. suis CCs than the PCR-RFLP method. The assay was capable of simultaneously distinguishing CC1, CC16, CC25, CC28, CC104, CC221/234, and CC233/379. PCR-RFLP could not clearly differentiate among most CCs in this study except CC16. DNA sequencing using the 16S-23S rDNA intergenic spacer distinguished between four clusters: 1) consisting of CC25, CC28, CC104, and CC233/379; 2) consisting of CC221/234; 3) consisting of CC16 (ST16); and 4) consisting of CC1. This study revealed that RAPD had a greater discriminatory power than PCR-RFLP. This assay will be useful for screening or predicting major CCs relevant to human and pig S. suis clinical isolates and for low-cost screening of large numbers of isolates with rapid analytical capacity and could be utilized in most laboratories.

The conservation landscape of the human ribosomal RNA gene repeats.

Ribosomal RNA gene repeats (rDNA) encode ribosomal RNA, a major component of ribosomes. Ribosome biogenesis is central to cellular metabolic regulation, and several diseases are associated with rDNA dysfunction, notably cancer, However, its highly repetitive nature has severely limited characterization of the elements responsible for rDNA function. Here we make use of phylogenetic footprinting to provide a comprehensive list of novel, potentially functional elements in the human rDNA. Complete rDNA sequences for six non-human primate species were constructed using de novo whole genome assemblies. These new sequences were used to determine the conservation profile of the human rDNA, revealing 49 conserved regions in the rDNA intergenic spacer (IGS). To provide insights into the potential roles of these conserved regions, the conservation profile was integrated with functional genomics datasets. We find two major zones that contain conserved elements characterised by enrichment of transcription-associated chromatin factors, and transcription. Conservation of some IGS transcripts in the apes underpins the potential functional significance of these transcripts and the elements controlling their expression. Our results characterize the conservation landscape of the human IGS and suggest that noncoding transcription and chromatin elements are conserved and important features of this unique genomic region.