Nucleotide sequence of theLeishmania donovanimedRNA gene

Abstract

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.

Lack of Abundance of Cytoplasmic Cyclosporin A-binding Protein Renders Free-living Leishmania donovaniResistant to Cyclosporin A

“…Thus, to characterize the transcript, an RT-PCR approach was adapted. Due to the phenomenon of transplicing in Leishmania, the 5Ј termini of all mature mRNAs are known to contain an identical 39-nucleotide-long mini-exon sequence (19). It was therefore reasoned that the use of this common mini-exon sequence as a sense primer along with the antisense internal oligonucleotide primer designed from the conserved CsA binding region (WLDGRHVVFG) should amplify at least one (LdCyP) if not all of the isoforms of the CyP gene family present in the total RNA.…”

“…The reaction was carried out at 37°C for 5 min followed by 1 h at 42°C. To amplify the LdCyP-specific cDNA, 20 pmol each of the sense primer, 5Ј-CTCGGAAT-TCCAACGCTATATAAGTATCAGTTTCTGTACTTTATTG-3Ј, containing a short leader sequence with an EcoRI site followed by 36 of the 39-nucleotide mini-exon (19), and an antisense primer, 5Ј-GCCGAAAACCA-CATGGCGG-3Ј (438 -456), designed from a part (GRHVVFG) of the conserved CsA binding motif (WLDGRHVVFG), were added to the reverse transcription mixture. The PCR reaction was continued for 35 cycles at 94, 55, and 68°C for 1, 1, and 2 min, respectively.…”

Glycoprotein 46 mRNA Abundance Is Post-transcriptionally Regulated during Development of Leishmania chagasiPromastigotes to an Infectious Form

“…Fourteen phage clones hybridized to the probe (0.05%), and 10 were plaque-purified for further characterization. The complete 2.4-kb sequence of the largest of these 10 cDNA inserts was determined and found to be a partial-length cDNA lacking the 39-nucleotide-spliced leader found on the 5Ј ends of all Leishmania mRNAs (32).…”

An Atypical Protein Disulfide Isomerase from the Protozoan Parasite Leishmania Containing a Single Thioredoxin-like Domain

“…The 5Ј-region of the gene was first amplified by reverse transcriptase PCR using total RNA isolated from L. donovani promastigotes as the template and the following 5Ј-and 3Ј-primers. The 5Ј-forward primer was based on the spliced leader sequence, which is added to all leishmanial mRNAs by transplicing (27). The 3Ј-reverse degenerate primer was deduced from a region containing the highly conserved active site -CGHC-motif of PDIs (8,9).…”

“…To identify putative PDI genes, this cDNA was used as a template in a polymerase chain reaction with the forward 5Ј-ACT AAC GCT ATA TAA GTA TCA-3Ј and reverse 5Ј-TT (A/G)CA (A/G)TG (A/G/C)CC (A/G)CA CCA-3Ј primers. The forward primer reflects a portion of the conserved Leishmania-specific mRNA splice leader sequence (27), and the degenerate reverse primer is based on conserved amino acids, including the active site, of various PDI sequences available in protein databases. The resulting PCR products were cloned into the pCRII cloning vector (Invitrogen) and sequenced.…”