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This report describes the complete translated gene sequence, predicted secondary structure and lipid bilayer association of a novel kinetoplastid membrane protein (KMP-11) from Leishmania donovani promastigotes. KMP-11 was previously referred to as the lipophosphoglycan-associated protein (LPGAP). The isolation, species distribution and chemical characterization, including a partial protein sequence analysis and post-translational modifications, of this major membrane component have been described [Jardim, Funk, Caprioli and Olafson (1995) Biochem. J. 305, 307-313]. C.d. measurements of KMP-11 indicated a very high helical content estimated to be approximately 86% in trifluoroethanol. This was in agreement with computer-based secondary-structure analyses which predicted KMP-11 to be almost exclusively alpha-helical, with the protein adopting a helix-loop-helix motif. Arrangement of the residues located in the putative helical regions on an Edmundson helical wheel showed that this molecule could have a strongly amphipathic conformation and provided an explanation for how such a highly charged protein might be inserted into the plasma membrane. Evidence in support of KMP-11 association with lipid bilayers was provided by showing that KMP-11 could mediate carboxyfluorescein release from liposomes. These findings suggested that KMP-11 may function in part to increase bilayer pressure, stabilizing molecules such as lipophosphoglycan within the parasite pellicular membrane.

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We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. cx-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

The trans-splicing reaction of kinetoplastid protozoa attaches a separately encoded 39 nucleotide mini-exon onto the 5' ends of all mRNAs (1). The presence of this defined sequence affords the possibility of facile mapping of the 5' termini of mature mRNAs in these primitive pathogens by amplification of RNA sequences using the polymerase chain reaction. The subcloning and sequencing of the resulting amplified DNAs circumvents the necessity of less accurate and more time consuming traditional methods for mapping 5' ends of transcripts, such as S I nuclease mapping or primer extension. Although the sequences of the miniexons in kineteoplastids are highly conserved (2), particularly among Leishmania species (3), only the first 15 nucleotides of the L. donovani mini-exon sequence have been reported.To develop a polymerase chain reaction based amplification system for examining the 5' termini of L. donovani mRNAs and to compare the sequences and components of the regions surrounding the mini-exon domain with that in other kinetoplastids, the L.donovani medRNA gene was cloned and a 429 bp ApaI fragment containing the complete medRNA transcript was sequenced in both directions (Figure 1). The 39 nucleotide miniexon sequence was identical to those previously described from other Leishmania species, whereas the spliced leader intron differed from that of L. enriettii in 4 nucleotide positions and contained a one nucleotide deletion. Outside the transcript regions, there was essentially no homology between the intergenic regions of the L. donovani and L. enriettii mini-exon gene repeats with the exception of an oligo dT tract following the 3' end of the medRNA (nucleotides 157-165), a feature which is also present in the mini-exon gene from Trypanosoma brucei (4) and Trypanosoma cruzi (5).To determine whether the medRNA gene of L. donovani is arranged in a tandem repeat, as in the L. enriettiigene, genomic DNA was partially digested with RsaI, an enzyme that cuts once in the ApaI fragment. Southern blots of RsaI digested DNA revealed the characteristic ladder of hybridization signals, each a multimer of the 429 bp unit, indicating that the mini-exon genes are arranged in a tandemly repeated array (Figure 2). This arrangement of the miniexon gene sequences in L. donovani was confirmed by complete digestion of genomic DNA with NcoI (Figure 2), ApaI, AvaI, and BglI, each of which excised a DNA fragment of 429 bp. Figure 1. Nucleotide sequence of the L.donovani medRNA gene and comparison with L enriettii medRNA gene (2). The mini-exon sequence is double underlined, while the intron is underlined once and the 3' end is indicated on the basis of the L.enrietii medRNA sequence.

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