Mini-exon derived RNA gene of Leishmania donovani: structure, organization and expression.

Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such as Leishmania which donates its 5'-terminal 39 nucleotides to the 5'-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene from Leishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5'-terminus of a cellular mRNA (β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5'- upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed.

Transcription and processing of ß-tubulin messenger RNA in Leishmania donovani promastigotes.

Transcription of the multicopy ß-tubulin locus in Leishmania donovani promastigotes was examined by nucleic acid hybridization techniques. By northern analysis of promastigote RNA multiple ß-tubulin mRNAs were detected. The major species of 2·2 kb RNA is derived from the tandem repeat cluster of ß-tubulin genes, the other two (2·4 and 2·6 kb) are presumably derived from dispersed genomic loci. Combined S1-nuclease and primer extension mapping experiments demonstrated the presence of a single 5'-terminus with a 35 nucleotide spliced-leader sequence. The 3'-termini are heterogeneous. The development of a nuclear run-on system in Leishmania for studying transcription of individual genes is reported. Active but transient RNA polymerase II activity was observed in this system. Using specific DNA probes for labelled run-on RNA it was shown that ß-tubulin transcription occurs asymmetrically (i.e., on one strand of the DNA template) in an α-amanitin sensitive manner. The significance of these results for the life cycle of the parasite is discussed.

Organization and chromosomal localization of ß-tubulin genes in Leishmania donovani.

The genomic organization and chromosomal location of the ß-tubulin isogenes in Leishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned ß-tubulin gene. We have cloned a ß-tubulin gene fragment, 3·3 kbp long, from genomic DNA of Leishmania donovani using a heterologous ß-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11-15 copies of the ß-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3·5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of the ß-tubulin clone and is found to be nearly identical to that of Leishmania mexicana amazonensis.