RESUMEN
The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle. To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2). The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes. We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies. Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S. pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect. However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature. Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery.
Asunto(s)
Ciclo Celular/fisiología , Plasmodium vivax/genética , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , Schizosaccharomyces/citología , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Quinasas CDC2-CDC28 , Clonación Molecular , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/fisiología , ADN Complementario/química , Regulación de la Expresión Génica , Biblioteca de Genes , Prueba de Complementación Genética , Humanos , Malaria Vivax/parasitología , Datos de Secuencia Molecular , Plasmodium vivax/citología , Proteínas Quinasas/química , Proteínas Protozoarias/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Here we describe the construction of a representative YAC library for the human malarial parasite Plasmodium vivax. As P. vivax cannot be maintained continuously under laboratory conditions, the P. vivax DNA necessary for the library construction was isolated from a single human patient presenting himself with vivax malaria to a local hospital in the Brazilian Amazon. Thus, this YAC library is the first of its kind to be generated from patient-derived material. The YAC library consists of 560 clones with an average insert size of 180 kb. Of 9 published P. vivax genes, 8 were found to be present in the library. In addition, 12 P. vivax telomeric YAC clones were identified.