Construction of trypanosome artificial mini-chromosomes

We report the preparation of two linear constructs which, when transformed into the procyclic form of Trypanosoma brucei, become stably inherited artificial mini-chromosomes. Both of the two constructs, one of 10 kb and the other of 13 kb, contain a T.brucel PARP promoter driving a chloramphenicol a...

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Bibliographic Details
Published in:Nucleic acids research 1995-12, Vol.23 (23), p.4893-4899
Main Authors: Lee, Mary Gwo-Shu, E, Yaping, Axelrod, Nancy
Format: Article
Language:English
Subjects:
Animals
Blotting, Southern
Chloramphenicol O-Acetyltransferase - genetics
Chromosome Mapping
Chromosomes - genetics
Cinnamates
DNA - analysis
DNA - genetics
Genetic Vectors - genetics
Hygromycin B - analogs & derivatives
Phosphotransferases - genetics
Promoter Regions, Genetic - genetics
Transformation, Genetic - genetics
Trypanosoma brucei
Trypanosoma brucei brucei - genetics
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Summary:We report the preparation of two linear constructs which, when transformed into the procyclic form of Trypanosoma brucei, become stably inherited artificial mini-chromosomes. Both of the two constructs, one of 10 kb and the other of 13 kb, contain a T.brucel PARP promoter driving a chloramphenicol acetyltransferase (CAT) gene. In the 10 kb construct the CAT gene is followed by one hygromycin phosphotransferase (Hph) gene, and in the 13 kb construct the CAT gene is followed by three tandemly linked Hph genes. At each end of these linear molecules are telomere repeats and subtelomeric sequences. Electroporation of these linear DNA constructs into the procyclic form of T.brucei generated hygromycin-B resistant cell lines. In these cell lines, the input DNA remained linear and bounded by the telomere ends, but it increased in size. In the cell lines generated by the 10 kb construct, the input DNA increased in size to 20–50 kb. In the cell lines generated by the 13 kb constructs, two sizes of linear DNAs containing the input plasmid were detected: one of 40–50 kb and the other of 150 kb. The increase in size was not the result of in vivo tandem repetitions of the input plasmid, but represented the addition of new sequences. These Hph containing linear DNA molecules were maintained stably in cell lines for at least 20 generations in the absence of drug selection and were subsequently referred to as trypanosome artificial mini-chromosomes, or TACs.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/23.23.4893