Clonado y caracterización de quinasas relacionadas con la CDC2 en el parásito Trypanosoma cruzi
Two CDC2-related protein kinase genes (crk), tzcrk3 and tzcrk1, were cloned from the parasite Trypanosoma cruzi. Recombinant proteins were used to raise specific antisera. Antibodies anti-TzCRK1recognized a protein of 33 kDa in the three forms of the parasite. This antiserumshowed that TzCRK1 is loc...
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| Formato: | Tesis doctoral publishedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales
1999
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| Acceso en línea: | https://hdl.handle.net/20.500.12110/tesis_n3128_Gomez https://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=aextesis&d=tesis_n3128_Gomez_oai |
| Aporte de: |
| Sumario: | Two CDC2-related protein kinase genes (crk), tzcrk3 and tzcrk1, were cloned from the parasite Trypanosoma cruzi. Recombinant proteins were used to raise specific antisera. Antibodies anti-TzCRK1recognized a protein of 33 kDa in the three forms of the parasite. This antiserumshowed that TzCRK1 is localized in the cytosol, kinetoplast and nucleus, and is constitutiverexpressed though the cell cycle. Purified anti-TzCRK1 IgGs, immunoprecipitated a kinaseactivity which phosphorylated histone H1 and Rb. The recombinant GST-TzCRK1 had high invitro kinase activity in the absense of regulatory proteins. CDC2 and CDKZ specific inhibitorscould not abolish the TzCRK1 kinase activity. Transfection of COS-7 cells with tzcrk1demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitatedwith cyclins E, D3 and A suggesting a role for this kinase in cell cyclecontrol. The TzCRK3 antisera recognized a 40 kDa protein in membrane and nuclear fractionsand a 54 kDa protein in the citosolic fraction. The p13SUC1 protein coprecipitated a kinaseactivity from the citosol of the epimatigote stage. Yeast complementation assays showed thatthese T. cruzi CRKs, could not rescue the yeast CDC28 function. The two hybrid system was used to identify and clone proteins that associate to TzCRK1. Threeclones were identified, which code for proteins with identity to cyclins from the PREG and PHo80 family. In other organisms, these cyclin like proteins are involved in the control ofphosphate metabolism. In S. cerevisiae, PH080 binds to the CDK protein PHO85, whichcontrols the transcription of the acid phosphatase gene and participates in the regulation of thecell cycle. These results suggest that TzCRK1 could be involved in cell cycle progression and/orin other processes such as phosphate metabolism. |
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