Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption.
Analysis of gene expression in Trypanosoma cruzi has been impeded by the lack of efficient, stable, DNA-mediated transfection systems. We describe here the establishment of such a system for T. cruzi. Stable transformants were isolated following integration of the circular transforming plasmid into the chromosome by homologous recombination. Mutants with a disrupted PUB12.5 polyubiquitin gene, resulting from targeted integration of the plasmid vector, have been isolated. A mutant harboring the disrupted PUB12.5 gene lacks the intact PUB12.5 mRNA as well as transcripts corresponding to the truncated gene. Genomic Southern-blot analysis indicates that the inserted plasmid is tandemly repeated in each of the clones analyzed. A secondary recombination event in one clone resulted in a deletion within the 2.65 calmodulin-ubiquitin locus, encompassing the sequence from the CalA2 calmodulin gene to the PUB12.5 polyubiquitin gene.
Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
S
S
Hariharan
J
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Ajioka
J
J
Swindle
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D010957:Plasmids; D011108:Polymers / Q000378:metabolism*; D025821:Polyubiquitin; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D014170:Transformation, Genetic; D014349:Trypanosoma cruzi / Q000235:genetics*; D014452:Ubiquitins / Q000235:genetics*
D016054:DNA, Protozoan; D011108:Polymers; D012333:RNA, Messenger; D016053:RNA, Protozoan; D014452:Ubiquitins; D025821:Polyubiquitin