Genetic Data Showing Evolutionary Links between Leishmania and Endotrypanum

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studiedrevealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

Dominant character of the molecular marker of a Biomphalaria tenagophila strain (Mollusca: Planorbidae) resistant to Schistosoma mansoni

Biomphalaria tenagophila population from Taim (state of Rio Grande do Sul, Brazil) is totally resistant toSchistosoma mansoni, and presents a molecular marker of 350 bp by polymerase chain reaction and restriction fragment length polymorphism of the entire rDNA internal transcriber spacer. The scope of this work was to determine the heritage pattern of this marker. A series of cross-breedings between B. tenagophila from Taim (resistant) and B. tenagophila from Joinville, state of Santa Catarina (susceptible) was carried out, and their descendants F1 and F2 were submitted to this technique. It was possible to demonstrate that the specific fragment from Taim is endowed with dominant character, since the obtained segregation was typically mendelian.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6%) asymptomatic and 17 acute (65.4%) including 5 (19.2%) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Genetic diversity among isolates of stemphylium solani from cotton

The fungus Stemphylium solani causes leaf blight of tomato (Lycopersicon esculentum) in Brazil. In recent years, severe epidemics of a new leaf blight of cotton (Gossipium hyrsutum) caused by S. solani occurred in three major cotton-growing Brazilian states (PR, MT and GO). Molecular analysis was performed to assess the genetic diversity among the S. solani isolates from cotton, and to verify their relationship with representative S. solani isolates from tomato. Random amplified polymorphic DNA (RAPD) markers and internal transcribed spacers of ribosomal DNA (rDNA) were used to compare 33 monosporic isolates of S. solani (28 from cotton and five from tomato). An isolate of Alternaria macrospora from cotton was also used for comparison. RAPD analysis showed the presence of polymorphism between the genera and the species. The A. macrospora and the S. solani isolates from cotton and tomato were distinct from each other, and fell into separate groups. Variation by geographic region was observed for the tomato isolates but not for the cotton isolates. Amplifications of the ITS region using the primer pair ITS4/ITS5 resulted in a single PCR product of approximately 600 bp for all the isolates. Similarly, when amplified fragments were digested with eight restriction enzymes, identical banding patterns were observed for all the isolates. Hence, rDNA analysis revealed no inter-generic or intra-specific variation. The genetic difference observed between the cotton and the tomato isolates provides evidence that S. solani attacking cotton in Brazil belongs to a distinct genotype.

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.

Phylogeny of South American Pogonieae (Orchidaceae, Vanilloideae) based on sequences of nuclear ribosomal (ITS) and chloroplast (psaB, rbcL, rps16, and trnL-F) DNA, with emphasis on Cleistes and discussion of biogeographic implications

Tribe Pogonieae (Orchidaceae), as Currently known, comprises live genera distributed from South to North America and Eastern Asia. Phylogenetic inferences within Cleistes and among genera of tribe Pogonieae were made based oil nrDNA (ITS) and cpDNA (trnL-F, rps16, rbcL, and psaB) Sequence data and maximum parsimony. Eighteen species of Cleistes, members of all other genera of Pogonieae, and outgroups were sampled. Analyses based oil individual DNA regions provided similar topologies. All evidence indicates that Cleistes is paraphyletic. The North American C. divaricata and C bifaria are more closely related to the temperate genera Isotria and Pogonia than to their Central and South American congeners, the latter Constituting a monophyletic group characterized by the production of nectar as reward, tuberous roots, and their distribution in Central and South America. The Amazonian Duckeella is sister to the remainder of Pogonieae. Taxonomic and biogeographic implications are discussed, and morphological synapomorphies are given For clades obtained in the inferred molecular phylogeny. (C) 2008 Gesellschaft fur Biologische Systematik. Published by Elsevier GmbH. All rights reserved.

Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

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

Identification of Rhizoctonia solani AG 1-IB in lettuce, AG 4 HG-I in tomato and melon, and AG 4 HG-III in broccoli and spinach, in Brazil

Fungi isolated in Brazil, from lettuce, broccoli, spinach, melon and tomato, were identified as Rhizoctonia solani. All lettuce isolates anastomosed with both AG 1-IA and IB subgroups and all isolates from broccoli, spinach, melon and tomato anastomosed with AG 4 subgroup HG-I, as well as with subgroups HG-II and HG-III. DNA sequence analyses of ribosomal internal transcribed spacers showed that isolates from lettuce were AG 1-IB, isolates from tomato and melon were AG 4 HG-I, and isolates from broccoli and spinach were AG 4 HG-III. The tomato isolates caused stem rot symptoms, the spinach, broccoli and melon isolates caused hypocotyl and root rot symptoms on the respective host plants and the lettuce isolates caused bottom rot. This is the first report on the occurrence in Brazil of R. solani AG 4 HG-I in tomato and melon, of AG 4 HG-III in broccoli and spinach and of AG 1-IB in lettuce.

Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes ?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units, inverted sequences, pseudogenes and chromosome loci

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish. Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were deter-mined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence in situ hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively, An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA. The presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements. Copyright (C) 2002 S. Karger AG, Basel.

Genetic identification of the sharks Rhizoprionodon porosus and R. lalandii by PCR-RFLP and nucleotide sequence analyses of 5S rDNA

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