RESUMO
A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.
Assuntos
Proteínas de Bactérias , DNA de Helmintos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Helminto/metabolismo , RNA de Helmintos/metabolismo , Schistosoma mansoni/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Helminto/química , Sondas de Oligonucleotídeos , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/citologia , Schistosoma mansoni/genética , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.
Assuntos
Metilação de DNA , DNA de Helmintos/metabolismo , Schistosoma mansoni/genética , Animais , Southern Blotting , Fragmentação do DNA , DNA de Helmintos/química , Feminino , Masculino , Mapeamento por Restrição , Schistosoma mansoni/metabolismoRESUMO
Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.
Assuntos
Proteínas de Ligação a DNA/genética , Schistosoma mansoni/genética , Animais , Sequência de Bases , Cromatografia de Afinidade , Proteínas do Ovo/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Genes de Helmintos/genética , Proteínas de Helminto/genética , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Nucleares/efeitos da radiação , Oligonucleotídeos/metabolismo , Oligonucleotídeos/efeitos da radiação , Oogênese/genética , Regiões Promotoras Genéticas/genética , Schistosoma mansoni/química , Raios UltravioletaRESUMO
Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.
Assuntos
Mitocôndrias Cardíacas/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Abelhas/enzimologia , Caenorhabditis elegans/enzimologia , Bovinos , Clonagem Molecular , Sequência Consenso , DNA Complementar , Complexo I de Transporte de Elétrons , Biblioteca Gênica , Humanos , Mitocôndrias Cardíacas/efeitos dos fármacos , Dados de Sequência Molecular , NADH NADPH Oxirredutases/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Receptores Androgênicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schistosoma mansoni/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Partículas Submitocôndricas/efeitos dos fármacos , Partículas Submitocôndricas/metabolismo , Testosterona/farmacologiaRESUMO
An HMG2-like protein was purified from nuclear extracts of adult Schistosoma mansoni. Investigation of the amino acid composition of the schistosome HMG2-like protein showed that glutamic acid, glycine, aspartic acid and lysine were the most abundant. Carbohydrate analysis showed that the HMG2-like protein presented a low degree of glycosylation, galactose or glucose being the major monosaccharide constituent. Incubation of live schistosomes with 32P followed by isolation of nuclear proteins showed that the HMG-2 like protein could be phosphorylated. Partial sequence analysis of cyanogen bromide peptides revealed the occurrence of a phosphorylation consensus motif. The schistosome HMG2-like protein was found to bind preferentially to single-stranded DNA. The results suggest that the major non-histone S. mansoni nuclear protein belongs to the HMG family.