Journal article
Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene
Molecular and biochemical parasitology, Vol.57(2), pp.317-330
1993
DOI: 10.1016/0166-6851(93)90207-E
PMID: 8381919
Abstract
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 (
NEO
r). A pBluescript-based vector was constructed in which the 0.9-kb
NEO
r 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clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by
NEO
r. 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.
Details
- Title: Subtitle
- Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene
- Creators
- Keiko Otsu - University of IowaJohn E Donelson - University of IowaLouis V Kirchhoff - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Molecular and biochemical parasitology, Vol.57(2), pp.317-330
- Publisher
- Elsevier B.V
- DOI
- 10.1016/0166-6851(93)90207-E
- PMID
- 8381919
- ISSN
- 0166-6851
- eISSN
- 1872-9428
- Language
- English
- Date published
- 1993
- Academic Unit
- Psychiatry; Infectious Diseases; Epidemiology; Biochemistry and Molecular Biology; Internal Medicine
- Record Identifier
- 9984280868802771
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