RESUMO
In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX(2)CX(4)HX(4)C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.
Assuntos
Núcleo Celular/metabolismo , Código Genético/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Dedos de Zinco/genética , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Coelhos , Trypanosoma cruzi/metabolismoRESUMO
In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.
Assuntos
Animais , Coelhos , Núcleo Celular/metabolismo , Código Genético/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Dedos de Zinco/genética , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
In order to standardize and to characterize a chondrocyte primary culture, cells from rat rib resting cartilage were used. High yield (0.99 +/- 0.18 x 10(6) cells/rat) and viability (91.76%) of costal cartilage cells was reached by enzymatic digestion with collagenase. The cells were cultivated in Dulbecco's medium (DME) supplemented with 10%. Heat inactivated newborn calf serum, at 37 degrees under humidified atmosphere of 5% CO2 in air. Two or three days after plating, the cells were attached to the surface of tissue culture weel, and began dividing. Adhesion was independent of plating density. The doubling time of cell population was found to be 23.19 hours. The cells became a monolayer and required easy maintenance. The results support the contention that rat costal cartilage is a good source of chondrocytes for primary culture cells experiments.
