RESUMEN
One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.
Asunto(s)
Genes Protozoarios/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Recombinación Genética/genética , Transformación Genética , Secuencia de Bases , Southern Blotting , Clonación Molecular , Biblioteca de Genes , Vectores Genéticos/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción/genética , Análisis de Secuencia de ADNRESUMEN
The yKu protein of Saccharomyces cerevisiae is important for genome stability by repressing recombination involving telomeric sequences. The mechanism of this repression is not known, but silent heterochromatin such as HML, HMR, and telomeres are compartmentalized at the nuclear periphery and yKu is proposed to interact with these regions and to play a role in telomeric silencing and tethering. We have utilized ChIP on chip, quantitative PCR, and quantitative recombination assays to analyze yKu binding and its effect on genome stability in wild-type and mutant backgrounds. Our data suggest that, although yKu binds to the TG1-3 repeats and other parts of the genome when needed, such as during nonhomologous end-joining, it specifically binds to core X sequences in addition to the mating-type loci, HML and HMR. Association with core X occurred in the absence of Sir proteins, and enhanced binding was observed at silenced ends compared to nonsilenced ends. In contrast, binding to HML and HMR was totally dependent on Sir2-4p and partially dependent on Sir1p with a stronger association at HML in both MATa and MATalpha strains. Using yku80 separation-of-function mutants, we show a direct correlation between core X binding and recombination rate. We believe our findings support our hypothesis that yKu and core X play a pivotal role in maintaining genome stability through nuclear architecture by mediating a defensive fold-back structure at yeast chromosome ends.