RESUMO
In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.
Assuntos
Animais , Humanos , Cafeína/farmacologia , Proteína Quinase C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Schistosoma mansoni/genética , Proteínas rho de Ligação ao GTP/genética , Genes de Helmintos , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Schistosoma mansoni/metabolismo , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The nematode parasite Ascaris lumbricoides infects the digestive tracts of over 1.4 billion people worldwide, and its sister species, Ascaris suum, has infected a countless number of domesticated and feral pigs. It is generally thought that the putative ancestor to these worms infected either humans or pigs, but with the advent of domestication, they had ample opportunity to jump to a new host and subsequently specialize and evolve into a new species. While nuclear DNA markers decisively separate the two populations, mitochondrial sequences reveal that three major haplotypes are found in A. suum and in A. lumbricoides, indicating either occasional hybridization, causing introgression of gene trees, or retention of polymorphism dating back to the original ancestral species. This article provides an illustration of the combined contribution of parasitology, archaeoparasitology, genetics and paleogenetics to the history of ascariasis. We specifically investigate the molecular history of ascariasis in humans by sequencing DNA from the eggs of Ascaris found among ancient archeological remains. The findings of this paleogenetic survey will explain whether the three mitochondrial haplotypes result from recent hybridization and introgression, due to intensive human-pig interaction, or whether their co-occurrence predates pig husbandry, perhaps dating back to the common ancestor. We hope to show how human-pig interaction has shaped the recent evolutionary history of this disease, perhaps revealing the identity of the ancestral host
Assuntos
Humanos , Animais , História Antiga , Ascaríase , Ascaris lumbricoides , Ascaris suum , Evolução Biológica , DNA Mitocondrial , Primers do DNA , Genes de Helmintos , Marcadores Genéticos , Haplótipos , Interações Hospedeiro-Parasita , Polimorfismo Genético , SuínosRESUMO
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
Assuntos
Animais , Expressão Gênica , RNA Mensageiro , Schistosoma mansoni , Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biblioteca Gênica , Genes de Helmintos , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Schistosoma mansoni , Transcrição GênicaRESUMO
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
Assuntos
Animais , Humanos , Proteínas de Helminto , Histonas , Proteínas Nucleares , Schistosoma mansoni , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Expressão Gênica , Biblioteca Gênica , Genes de Helmintos , Proteínas Heterotriméricas de Ligação ao GTP , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Schistosoma mansoniRESUMO
The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1
Assuntos
Animais , Clonagem Molecular , Genes de Helmintos/genética , Proteínas de Helminto/genética , Schistosoma mansoni/genética , Dedos de Zinco/genética , Sequência de Bases , DNA Complementar , Proteínas de Ligação a DNA , Amplificação de Genes , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Genes de Helmintos/fisiologia , Genoma Bacteriano , Reação em Cadeia da PolimeraseRESUMO
We have established an in vitro culture system for adult schistosomes that allows monitoring gene expression for up to more than ten days. Comparing female worms that are paired with those that have been separated, we find distinct differences, clearly documenting an influence of the male in female gene expression. In perfect coincidence with classical observations that were based on histological techniques, we find that the male particularly regulates gene expression in those tissues that are characterized by cell proliferation, e.g. the vitellaria. From these results, we hypothesize that the key target for the inductive signal that is transferred from the male to the female during pairing is the activation of a growth factor that stimulates mitotic proliferation.
Assuntos
Animais , Cricetinae , Feminino , Masculino , Schistosoma mansoni , Northern Blotting , DNA de Helmintos , Fertilidade , Regulação da Expressão Gênica , Genes de Helmintos , Schistosoma mansoni , Fatores de Tempo , Transdução de SinaisRESUMO
Incubation of total protein extracts of Schistosoma mansoni with 3H 17-beta-estradiol and 20-hydroxyecdysone, revealed steroid binding proteins in both, male and female worms. The interaction of nuclear proteins with restriction fragments of the gender and stage-specific gene F-10 was investigated using the "Band-Shift" technique. Distinct male and female nuclear proteins bound to the fragments of this gene. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the estrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females, a band profile similar to that obtained with male worm protein extracts. When Tamoxifen was injected into schistosome infected mice, the eggs produced by females presented an abnormal morphology, compatible with non-viable eggs. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.
Assuntos
Animais , Masculino , Feminino , Cricetinae , Proteínas de Ligação a DNA , Proteínas de Helminto/metabolismo , Proteínas do Ovo/metabolismo , Schistosoma mansoni , Caracteres Sexuais , Genes de Helmintos , Ligação Proteica , Proteínas Nucleares/metabolismo , Schistosoma mansoniRESUMO
Genetic crosses between phenotypically resistant and sensitive schistosomes demonstrated that resistance to hycanthone and oxamniquine behaves like a recessive trait, thus suggesting that resistance is due to the lack of some factor. We hypothesized that, in order to kill schistosomes, hycanthone and oxamniquine need to be converted into an active metabolite by some parasite enzyme wich, if inactive, results in drug resistance. Esterification of the drugs seemed to be the most likely event as it would lead to the production of an alkylating agent upon dissociation of the ester. An artificial ester of hycanthone was indeed active even in resistant worms, thus indirectly supporting our hypothesis. In addition, several lines of evidence demonstrated that exposure to hycanthone and oxamniquine results in alkylation of worm macromolecules. Thus, radioactive drugs formed covalent bonds with the DNA of sensitive (but not of resistant) schistosomes; an antiserum raised against hycanthone detected the presence of the drug in the purified DNA fraction of sensitive (but not of resistant) schistosomes; a drug-DNA adduct was isolated from hycanthone-treated worms and fully characterized as hycanthone-deoxyguanosine.