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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Three sympatric karyomorphs in the fish Astyanax fasciatus (Teleostei, Characidae) do not seem to hybridize in natural populations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chromosomal mapping of repetitive DNAs in the beetle Dichotomius geminatus provides the first evidence for an association of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity between the B chromosome and A complement

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phylogenetic relationships in genus Arachis based on ITS and 5.8S rDNA sequences

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chromosomal location of heterochromatin and 45S rDNA sites in four South American triatomines (Heteroptera: Reduviidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of Rhizoctonia solani associated with soybean in Brazil by rDNA-ITS sequences

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

Intraspecific evolution of Rhizoctonia solani AG-1 IA associated with soybean and rice in Brazil based on polymorphisms at the ITS-5.8S rDNA operon

Rhizoctonia solani AG-1 IA causes leaf blight on soybean and rice. Despite the fact that R. solani AG-1 IA is a major pathogen affecting soybean and rice in Brazil and elsewhere in the world, little information is available on its genetic diversity and evolution. This study was an attempt to reveal the origin, and the patterns of movement and amplification of epidemiologically significant genotypes of R. solani AG-1 IA from soybean and rice in Brazil. For inferring intraspecific evolution of R. solani AG-1 IA sampled from soybean and rice, networks of ITS-5.8S rDNA sequencing haplotypes were built using the statistical parsimony algorithm from Clement et al. (2000) Molecular Ecology 9: 1657-1660. Higher haplotype diversity (Nei M 1987, Molecular Evolutionary Genetics Columbia University Press, New york: 512p.) was observed for the Brazilian soybean sample of R. solani AG-1 IA (0.827) in comparison with the rest of the world sample (0.431). Within the south-central American clade (3-2), four haplotypes of R. solani AG-1 IA from Mato Grosso, one from Tocantins, one from Maranhao, and one from Cuba occupied the tips of the network, indicating recent origin. The putative ancestral haplotypes had probably originated either from Mato Grosso or Maranhao States. While 16 distinct haplotypes were found in a sample of 32 soybean isolates of the pathogen, the entire rice sample (n=20) was represented by a single haplotype (haplotype 5), with a worldwide distribution. The results from nested-cladistic analysis indicated restricted gene flow with isolation by distance (or restricted dispersal by distance in nonsexual species) for the south-central American clade (3-2), mainly composed by soybean haplotypes.

EVOLUTION OF ADENINE CLUSTERING IN 5S RIBOSOMAL-RNA

The frequency of adenine mononucleotides (A), dinucleotides (AA) and clusters, and the positions of clusters, were studied in 502 molecules of the 5S rRNA.All frequencies were reduced in the evolutive lines of vertebrates, plants and fungi, in parallel with increasing organismic complexity. No change was observed in invertebrates. All frequencies were increased in mitochondria, plastids and mycoplasmas. The presumed relatives to the ancestors of the organelles, Rhodobacteria alfa and Cyanobacteria, showed intermediate values, relative to the eubacterial averages. Firmibacterid showed very high number of cluster sites.Clusters were more frequent in single-stranded regions in all organisms. The routes of organelles and mycoplasmas accummulated clusters at faster rates in double-stranded regions. Rates of change were higher for AA and clusters than for A in plants, vertebrates and organeltes, higher for cluster sites and A in mycoplasmas, and higher for AA and A in fungi. These data indicated that selection pressures acted more strongly on adenine clustering than on adenine frequency.It is proposed that AA and clusters, as sites of lower informational content. have the property of tolerating positional variation in the sites of other molecules (or other regions of the same molecule) that interact with the adenines. This reasoning was consistent with the degrees of genic polymorphism. low in plants and vertebrates and high in invertebrates. In the eubacteria endosymbiontic or parasitic to eukaryotes, the more tolerant RNA would be better adapted to interactions with the homologous nucleus-derived ribosomal proteins: the intermediate values observed in their precursors were interpreted as preadaptive.Among other groups, only the Deinococcus-Thermus eubacteria showed excessive AA and cluster contents, possibly related to their peculiar tolerance to mutagens, and the Ciliates showed excessive AA contents, indicative of retention of primitive characters.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65(.)2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10(.)9 %), Spirochaetes (4(.)3 %), Bacteroidetes (6(.)5 %), Actinobacteria (2(.)2 %) and Deferribacteres (2(.)2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.

Molecular analysis of the rDNA ITS-1 intergenic spacer in Drosophila mulleri, D-arizonae, and their hybrids

We analyzed the ITS-1 spacer region of the rDNA in Drosophila mulleri and D. arizonae, two sibling species belonging to the mulleri complex (repleta group) and in hybrids obtained in both cross directions. In spite of several previous studies showing the incompatibility of crosses involving D. arizonae females and D. mulleri males, we were able to obtain hybrids in this direction. Complete ITS-1 region was amplified using primers with homology at the 3'-end of the 18S rDNA and the 5'-end of the 5.8S rDNA genes. Our data demonstrated that D. mulleri and D. arizonae can be differentiated as they present a difference in length for the ITS-1 region. The amplified fragment for this region in D. mulleri has a length of 600 bp, whereas in D. arizonae this fragment is about 500 bp. It was also observed that male and female hybrids obtained in both cross directions present two amplified fragments, confirming the location of the ribosomal cistrons in the X chromosomes and microchromosomes of both parental species.

rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil

In this paper we present the first report of the occurrence of a binucleate Rhizoctonia spp. causing hypocotyl and root rot in kale in Brazil. Rhizoctonia spp. were isolated from kale (Brassica oleracea var. acephala) with symptoms of hypocotyl and root rot. The isolates, characterized as binucleate Rhizoctonia spp., did not show an anastomosis reaction with any of the binucleate Rhizoctonia spp. testers used. The pathogenicity of the isolates was tested under greenhouse conditions; all isolates were pathogenic and showed different symptom severities on kale. The ITS-5.8S rDNA sequences of kale isolates and 50 testers (25 binucleate Rhizoctonia spp. and 25 Rhizoctonia solani) were compared in order to characterize the genetic identity of Rhizoctonia spp. infecting kale. The kale isolates showed genetic identities ranging from 99.3 to 99.8% and were phylogenetically closely related to CAG 7 (AF354084), with identities of 98.5 and 98.7%. It is suggested that the binucleate Rhizoctonia spp. causing hypocotyl and root rot on kale Brazil comprises a new AG not yet described.