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.
Otsu
K
K
Department of Internal Medicine, University of Iowa, Iowa City, USA.
Donelson
J E
JE
Kirchhoff
L V
LV
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