Trypanosoma cruzi: interruption of both alleles of a gene encoding a protein containing 14-amino-acid repeats by targeted insertion of NEOr and HYGr.
In Trypanosoma cruzi approximately 90% of the 121- and 176-kDa cytoskeletal proteins encoded by the two alleles of the TCR27 gene is composed of 14-amino-acid repeats. To gain insight into the function of the TCR27 proteins we replaced the corresponding regions of 42-nucleotide repeats in the two alleles with the NEOr and HYGr genes. Analyses of DNAs and RNAs from four clones resistant to both G418, a neomycin analogue, and hygromycin showed that in both cases the repetitive regions had in fact been deleted. In addition, the absence of expression of the 14-amino-acid repeats was confirmed in Western blots. In axenic cultures growth rates of the morphologically unchanged, doubly resistant organisms were not different from those of wild-type parasites. However, the doubly resistant organisms proliferated more slowly in cultured mammalian cells than did wild-type parasites. These findings indicate that the absence of the TCR27 repetitive regions is detrimental, but not fatal, to the parasites.
Department of Internal Medicine, University of Iowa, Iowa City, USA.
K
K
Otsu
J E
JE
Donelson
L V
LV
Kirchhoff
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D006921:Hygromycin B / Q000494:pharmacology; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009355:Neomycin / Q000494:pharmacology; D015800:Protozoan Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014349:Trypanosoma cruzi / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D014407:Tumor Cells, Cultured
D003598:Cytoskeletal Proteins; D015800:Protozoan Proteins; D006921:Hygromycin B; D009355:Neomycin