Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene.
In Trypanosoma cruzi, the cause of Chagas' disease in Latin America, a large proportion of the antigenic proteins described to date have repetitive domains. In earlier work we identified a partial length cDNA, designated TCR27, encoding approx. 26 copies of a 14-amino acid repeat and a unique 61-amino acid C-terminal region. The goal of the current project was to replace the repetitive region of a TCR27 gene with the neomycin phosphotransferase gene (NEOr). A pBluescript-based vector was constructed in which the 0.9-kb NEOr coding region replaced the 2.9-kb internal repetitive segment of a TCR27 gene and was in frame with its nonrepetitive 5' coding sequence (pTCR27-2::NEO). Epimastigotes were electroporated in the presence of linearized pTCR27-2::NEO and transfected clones were selected on solid medium containing G418. Southern and Northern analyses of DNAs and RNAs from four G418-resistant clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by NEOr. The absence of the native TCR27-2 protein in the transfected clones was confirmed by Western blot. In axenic cultures growth rates of epimastigotes bearing an interrupted TCR27-2 gene were not different from those of wild-type parasites. In addition, there was no relative impairment of the four transfected clones' ability to proliferate in cultured mammalian cells. The fact that the clones having the interrupted TCR27-2 gene were not impaired biologically suggests that the length of the repetitive region of the TCR27 protein is not a critical factor for survival.
Otsu
K
K
Department of Internal Medicine, University of Iowa, Iowa City 52242.
Donelson
J E
JE
Kirchhoff
L V
LV
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014349:Trypanosoma cruzi / Q000235:genetics*
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D010770:Phosphotransferases; D019868:Kanamycin Kinase