Mitochondrial minicircle DNA supports plasmid replication and maintenance in nuclei of Trypanosoma brucei.

Abstract

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)

tRNAs are imported into mitochondria of Trypanosoma brucei independently of their genomic context and genetic origin.

“…Since one aim of this study was to test whether 5'and 3'-flanking sequences of defined lengths are involved in import, genomic integration would not be optimal, as influences of sequences upstream or downstream of the transfected gene cannot be excluded. We therefore decided to express the tRNAs episomally using the pTho plasmid (Metzenberg and Agabian, 1994). pTho contains a PARP (procyclic acidic repetitive protein)/ procyclin promoter/hygromycin resistance gene cassette followed by a minicircle sequence of 1 kb in length ( Figure IA).…”

“…Recombinant plasmids pTho-1 (generously provided by Drs Stan Metzenberg and Nina Agabian), which due to the minicircle sequence is maintained and replicated as an episome in Tbrucei (Metzenberg and Agabian, 1994), was used as a vector for the transfection experiments. The plasmid was modified by inserting a synthetic sequence, containing a unique BglI site, immediately downstream of the unique KpnI site located 5' of the PARP/procyclin promoter.…”

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

“…A recently developed extrachromosomally maintained vector for the transformation of Tbrucei (Metzenberg and Agabian, 1994) will allow us to examine the effects of mutating both the SLA2 RNA gene and a 3' splice site reporter construct. The remaining U5 RNA-RNA interactions occur at the 3' splice sites, which are diverse in Tbrucei as in other organisms.…”

“…Since the SLA2 RNA is present as a single-copy gene in Tbrucei (W.P.Mitchell, J.Holmes, J.M.Dungan and N.Agabian, unpublished observations), we should be able to determine whether it is essential and to dissect its role(s) in transsplicing genetically. A recently developed extrachromosomally maintained vector for the transformation of Tbrucei (Metzenberg and Agabian, 1994) will allow us to examine the effects of mutating both the SLA2 RNA gene and a 3' splice site reporter construct.…”

Construction of trypanosome artificial mini-chromosomes

“…The efficiency of this type of transformation can be as high as 1 x 1i-3, as has been calculated by using the tubulin locus as a target site (9). Secondly, to avoid detrimental effects or mutations at a specific chromosomal location, several plasmid DNA constructs have been created which allow the formation of extrachromosomal circular DNA molecules (episomes) in transformed trypanosomes and other trypanosomatids (10)(11)(12)(13)(14)(15). One type of these plasmids, once inside the cell, forms large episomes containing tandemly linked input plasmid.…”