In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a m ondrial kinetlst DNA (kDNA) micircie of Trypanosoma and conserved sequence characteristics such as purine/ pyrimidine strand biases. A specific UMS-binding protein has been identified in kinetoplastids (9); however, it is not known whether it has a role in kDNA replication.It has recently become possible to transfect Trypanosoma brucei with either transient expression vectors or through homologous recombination (10); however, we wished to increase the genetic capabilities of the organism by developing a shuttle vector that would allow the maintenance of multiple extrachromosomal copies ofa transfected sequence.By introducing random fiagments oftotal T. brucei DNA into a vector carrying a hygromycin-resistance gene, a mitochondrial DNA element was identifiedt that permits plasmid replication and maintenance in the nuclei of T. brucei. The plasmid is maintained stably under continuous drug selection as a supercoiled head-to-tail concatemer composed of approximately eight monomer units. A second kDNA minicircle element, chosen at random and similarly tested, also permits autonomous replication. These findings suggest that minicircles may have the interesting capability of engendering DNA replication in both the mitochondrion and nucleus.
MATERIALS AND METHODSTrnfection of Cels. T. brucei (subspecies brucei, strain IsTat 1.1) were cultured in Cunningham's medium (11) supplemented with 10%o (vol/vol) fetal bovine serum. Electroporation and transfection of T. brucei were performed by the method ofKapler (12) with 10-50 pg ofplasmid DNA isolated from Escherichia coli (SURE strain, Stratagene) by Qiagen (Chatsworth, CA) column chromatography. After electroporation, cells were cultured for 24-48 hr without selection before the addition of 100 pg of hygromycin B (Calbiochem) per ml.Purification of DNA from T. brucei. Total DNA was prepared from T. bracei by the method of Milhausen et al. (13) or White et al. (14) and dialyzed against TE (10 mM Tris chloride/i mM EDTA, pH 7.5). Supercoiled DNA molecules were fractionated by equilibrium centrifugation (15)
