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PubMed1989

Mapping of ribosomal RNA transcripts in wheat.

Ribosomal RNA transcripts in wheat have been studied by RNA gel blotting and their termini determined from electrophoretic analysis of S1 nuclease-resistant RNA/DNA hybrids and also of hybrids created by primer extension. A major putative transcription initiation site has been localized 1132 base pairs upstream from the 5' end of the 18S RNA sequence. A major putative processing site occurs 640 base pairs from the 5' end of the 18S RNA. Transcripts extending at least 750 base pairs beyond the 3' end of the 25S rRNA sequence into the array of intergenic repeats are present, as are transcripts covering the principal initiation sequence. This suggests that some transcripts extend through the intergenic DNA from one repeat unit into the next.

Base Sequence↗
PubMed2002

Speciation in ancient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis (Rotifera).

Continental lake-dwelling zooplanktonic organisms have long been considered cosmopolitan species with little geographic variation in spite of the isolation of their habitats. Evidence of morphological cohesiveness and high dispersal capabilities support this interpretation. However, this view has been challenged recently as many such species have been shown either to comprise cryptic species complexes or to exhibit marked population genetic differentiation and strong phylogeographic structuring at a regional scale. Here we investigate the molecular phylogeny of the cosmopolitan passively dispersing rotifer Brachionus plicatilis (Rotifera: Monogononta) species complex using nucleotide sequence variation from both nuclear (ribosomal internal transcribed spacer 1, ITS1) and mitochondrial (cytochrome c oxidase subunit I, COI) genes. Analysis of rotifer resting eggs from 27 salt lakes in the Iberian Peninsula plus lakes from four continents revealed nine genetically divergent lineages. The high level of sequence divergence, absence of hybridization, and extensive sympatry observed support the specific status of these lineages. Sequence divergence estimates indicate that the B. plicatilis complex began diversifying many millions of years ago, yet has showed relatively high levels of morphological stasis. We discuss these results in relation to the ecology and genetics of aquatic invertebrates possessing dispersive resting propagules and address the apparent contradiction between zooplanktonic population structure and their morphological stasis.

Animals↗
PubMed2005

Prevalence of tinea capitis in Ethiopian schoolchildren.

The prevalence of dermatophytosis and the spectrum of dermatophyte species were determined in children attending two schools in Addis Ababa, Ethiopia. Demographic and clinico-dermatological data were collected. Specimens were taken for microscopy and culture from all suspected lesions. Dermatophyte species were identified by morphology and biochemical tests, supplemented by sequencing of the rDNA ITS 2 region in selected isolates. From the Biruh Tesfa Elementary School (BTES) 824 students, and from Mount Olive Academy (MOA) all 124 students, were included. In BTES 513 (62.3%) students were clinically diagnosed with dermatophytosis, 463 (90.3 %) of them with tinea capitis. In 200 consecutive samples from BTES, and in 66 from MOA, 75 and 62%, respectively, contained fungal elements at microscopy. From BTES, 163/496 (33%) samples were culture-positive, of which 149 (91.4%) grew with dark purple colonies identified as Trichophyton violaceum, while 244 (49.4%) samples were contaminated. A few strains grew slowly developing white to cream colonies, two were identified as T. verrucosum, and 12 as white T. violaceum. From MOA 44 (66.7%) of samples were culture-positive, 38 (87%) were identified as T. violaceum, and one (2.3%) as T. verrucosum, while 33% showed no growth. Four white isolates of T.violaceum were confirmed by DNA-sequencing. Dermatophytosis was thus diagnosed in 55-62% of children screened at two schools of different socioeconomic standards in the Ethiopian capital. Trichophyton violaceum constituted 87-90% of all isolates. White variants of T. violaceum were diagnosed in 16 cases.

Adolescent↗
PubMed2006

The lichen-forming ascomycete Evernia mesomorpha associates with multiple genotypes of Trebouxia jamesii.

The epiphyte Evernia mesomorpha forms a lichen association with green algae in the genus Trebouxia. Little is known about the population structure of E. mesomorpha. Here, population structure of the algal and fungal symbionts was examined for 290 lichen thalli on 29 jack pine (Pinus banksiana) trees in Manitoba. Through phylogenetic analysis of internal transcribed spacer (ITS) nuclear ribosomal DNA (rDNA) sequences, five algal genotypes were detected that were nested within T. jamesii. Two fungal genotypes were detected that formed a clade with two other Evernia species. The genus Evernia was paraphyletic with E. prunastri, sister to Parmelia saxatilis. Restriction fragment length polymorphism (RFLP) of ITS rDNA showed multiple algal genotypes in 45% of the 290 lichen thalli collected, whereas all thalli only contained one fungal genotype. Low population subdivision of algal and fungal genotypes among trees suggested that the algal symbiont was being dispersed in the lichen soredium. Low fungal specificity for multiple algal genotypes and a hypothesized algal switch may be important life history strategies for E. mesomorpha to adapt to changing environmental conditions.

Ascomycota↗
PubMed2006

Reciprocal hybridization at different times between Senecio flavus and Senecio glaucus gave rise to two polyploid species in north Africa and south-west Asia.

The analysis of hybrid plant taxa using molecular methods has considerably extended understanding of possible pathways of hybrid evolution. Here, we investigated the origin of the tetraploid Senecio mohavensis ssp. breviflorus and the hexaploid Senecio hoggariensis by sequencing of nuclear and chloroplast DNA, and by analysis of the distribution of taxon-specific amplified fragment length polymorphism (AFLP) fragments. Both taxa originated from hybridization between the diploid Senecio flavus and Senecio glaucus. Whereas S. glaucus was the female parent in the origin of S. mohavensis ssp. breviflorus, S. flavus was the female parent in the origin of S. hoggariensis. The distribution of AFLP fragments suggests that S. hoggariensis is an allohexaploid species with two diploid genomes of S. glaucus and one diploid genome of S. flavus. The high frequency of S. flavus-specific fragments in S. mohavensis ssp. breviflorus is explained either as the result of introgression between a primary hybrid and S. flavus or as the result of intergenomic recombination in a primary hybrid. These two alternative processes cannot easily be distinguished.

Africa↗
PubMedDate not supplied

Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus.

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.

Animals↗
PubMedDate not supplied

Development and molecular characterisation of the microsporidian Schroedera airthreyi n. sp. in a freshwater bryozoan Plumatella sp. (Bryozoa: Phylactolaemata).

The development of a new species of microsporidian, infecting a freshwater Plumatellid bryozoan, is described. The small-subunit rDNA, internal transcribed spacer region (ITS), and partial large-subunit rDNA genes were sequenced. Phylogenetic analysis demonstrated that the parasite clustered with Schroedera plumatellae. However, while there were morphological affinities with this species, significant differences were also observed. The infection initially appeared as a roughening of the peritoneum lining the metacoelom of the bryozoan. This roughening resolved into meront-infected syncytia, composed of interconnected cells of the body wall that detached to float in the coleomic cavity. Spores were observed to develop within these syncytia. All stages of development were diplokaryotic in contrast to S. plumatellae, which has a distinct monokaryotic merogony preceding sporogony. The infection was pathogenic to the host. Direct bryozoan-bryozoan transmission was not observed. We propose to name the microsporidian Schroedera aithreyi n. sp.

Animals↗
PubMed2000

Saccharomyces uvarum, a proper species within Saccharomyces sensu stricto.

Saccharomyces uvarum is proposed as a proper species within the complex Saccharomyces sensu stricto. Molecular characteristics including the similarity of the restriction profile of the non-transcribed spacer 2 (NTS2) and of the D1/D2 sequences of the rDNA, as well as other genotypic and phenotypic characteristics confirm that this group of strains is highly homogeneous and distinguishable from other species of the Saccharomyces sensu stricto group.

DNA, Bacterial↗
PubMed2001

Molecular mechanism of MexR-mediated regulation of MexAB-OprM efflux pump expression in Pseudomonas aeruginosa.

To elucidate the molecular mechanism by which the MexR repressor regulates expression of the MexAB-OprM efflux pump, we investigated MexR and the mexR-mexA intergenic DNA (mexOP) interaction, and transcription of the mexA-lacZ reporter gene containing different lengths of mexOP. Homogeneously purified MexR bound specifically to mexOP proximal to mexR. The mexOP-lacZ fusion gene lacking the region immediately proximal to mexR showed minimum enzyme activity, thereby suggesting that a promoter element is located between mexR and the MexR-binding sites. These observations explain the mechanism of self-regulation of mexR expression as well as low and elevated expression of MexAB-OprM in the wild-type strain and nalB-type mutant, respectively, of Pseudomonas aeruginosa.

Bacterial Outer Membrane Proteins↗
PubMed2002

Microbial composition of near-boiling silica-depositing thermal springs throughout Yellowstone National Park.

The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences.

Archaea↗
PubMed2002

Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer.

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

Animals↗
PubMed2003

Electrophoretic analysis of genetic variability within Cryptosporidium parvum from imported and autochthonous cases of human cryptosporidiosis in the United Kingdom.

Cryptosporidium parvum oocyst DNA samples (n = 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (approximately 300 bp) and second internal transcribed spacer (pITS-2) (approximately 230 bp) of nuclear ribosomal DNA. The two recognized genotypes (types 1 and 2) of C. parvum could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of approximately 1.3 to 1.7%. Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as C. parvum type 1 and 14 (13.7%) were identified as type 2. For outbreak samples, unequivocal differentiation between type 1 (n = 20; one child nursery outbreak) and type 2 (n = 62; two waterborne outbreaks) was also achieved. Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of approximately 1 to 42%) than that in pSSU. SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source. Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis. The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks.

Animals↗
PubMed2005

First detection and genotyping of human-associated microsporidia in pigeons from urban parks.

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.

Animals↗
PubMed2004

Transcription initiation and termination on Leishmania major chromosome 3.

Genome projects involving Leishmania and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire Leishmania major Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the "left" telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the "right" telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.

Amanitins↗
PubMed1997

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.

Bacteriophages↗
PubMed2003

Genetic Heterogeneity in the rRNA Gene Locus of Trichophyton tonsurans.

Trichophyton tonsurans is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of T. tonsurans by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated T. tonsurans from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in T. tonsurans.

Base Sequence↗
PubMed2003

Rapid identification of the genus fonsecaea by PCR with specific oligonucleotide primers.

An oligonucleotide primer set based on internal transcribed spacer regions of ribosomal DNA for PCR which gives the amplicon for only the DNA from Fonsecaea species was designed. This set yielded an amplicon with 333 bp for all strains of Fonsecaea pedrosoi and Fonsecaea compacta examined but no amplicons for related dematiaceous fungi and pathogenic yeasts. PCR using this primer set was considered to be a useful method for the rapid identification of the genus FONSECAEA:

Ascomycota↗