Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

Future trypanosomatid phylogenies: refined homologies, supertrees and networks

There has been good progress in inferring the evolutionary relationships within trypanosomes from DNA data as until relatively recently, many relationships have remained rather speculative. Ongoing molecular studies have provided data that have adequately shown Trypanosoma to be monophyletic and, rather surprisingly, that there are sharply contrasting levels of genetic variation within and between the major trypanosomatid groups. There are still, however, areas of research that could benefit from further development and resolution that broadly fall upon three questions. Are the current statements of evolutionary homology within ribosomal small sub-unit genes in need of refinement? Can the published phylograms be expanded upon to form `supertrees' depicting further relationships? Does a bifurcating tree structure impose an untenable dogma upon trypanosomatid phylogeny where hybridisation or reticulate evolutionary steps have played a part? This article briefly addresses these three questions and, in so doing, hopes to stimulate further interest in the molecular evolution of the group.

Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V.) peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V.) braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus

Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.

Courtship song genes and speciation in sand flies

Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) is a vector of visceral leishmaniasis in the Americas and it might represent a complex of sibling species. Reproductive isolation between closely related species often involves differences in courtship behaviour. cacophony (cac) and period (per) are two Drosophila genes that control features of the "lovesong" males produce during courtship that has been implicated in the sexual isolation between closely related species. We are using gene fragments from L. longipalpis' homologues of these two genes to study the speciation process in this putative species complex.

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.

Clustering of Schistosoma mansoni mRNA sequences and analysis of the most transcribed genes: implications in metabolism and biology of different developmental stages

The study of the Schistosoma mansoni genome, one of the etiologic agents of human schistosomiasis, is essential for a better understanding of the biology and development of this parasite. In order to get an overview of all S. mansoni catalogued gene sequences, we performed a clustering analysis of the parasite mRNA sequences available in public databases. This was made using softwares PHRAP and CAP3. The consensus sequences, generated after the alignment of cluster constituent sequences, allowed the identification by database homology searches of the most expressed genes in the worm. We analyzed these genes and looked for a correlation between their high expression and parasite metabolism and biology. We observed that the majority of these genes is related to the maintenance of basic cell functions, encoding genes whose products are related to the cytoskeleton, intracellular transport and energy metabolism. Evidences are presented here that genes for aerobic energy metabolism are expressed in all the developmental stages analyzed. Some of the most expressed genes could not be identified by homology searches and may have some specific functions in the parasite.

Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil

Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified. This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H. pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil. Gastric biopsy of 61 H. pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction. Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT). The prevalence of H. pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p = 0.02). The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2. However, the prevalence of the vacA s1 genotying was the same in either DU or GT group. The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b. In resume there was a strong association between the H. pylori cagA+ gene and DU. However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominat.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

A ribosome association factor (AF) was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-Bromoindolenine (BNPS-skatole). This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.

Evaluation of adherence, hemagglutination, and presence of genes codifying for virulence factors of Acinetobacter baumannii causing urinary tract infection

Acinetobacter baumannii is a strictly aerobic bacterium which causes severe infections, however its pathogenic characteristics are not well defined. Thirteen A. baumannii strains isolated from urine of hospitalized and nonhospitalized patients with different ages were investigated for the presence of virulence factors. The isolates belonged to biotypes 2, 6, and 9 and were sensitive to imipenem. The majority of them showed resistance to amikacin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, norfloxacin, and trimethoprim-sulfamethoxazole. None of A. baumannii strains presented genes codifying for 17 different virulence factors previously described in uropathogenic Escherichia coli, when tested by polymerase chain reaction (PCR). Nine isolates agglutinated human group AB erythrocytes, in presence of mannose, but none of them agglutinated group O erythrocytes. Adherence to polystyrene was observed in 7 isolates, and this result did not correlate with that obtained in hemagglutination assay. All the isolates were able to grow in iron-limiting conditions, showing that A. baumannii produces some type of siderophore. However, the genes iutA and fyuA, from iron uptake system of E. coli and Yersinia sp., respectively, were not present in the isolates, suggesting the presence of a different type of siderophore. The fimbriae of A. baumannii strains that mediates the adherence are possibly mannose-resistant, eventhough the mechanism of adherence to human epithelial cells still remains to be elucidated.