Résumé
The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were obtained. Reduction of the gp63 level resulted in increased generation times, altered cell morphology, accumulation of cells in the G2/M phase of the cell cycle, and increased numbers of binucleate cells with one or two kinetoplasts. Growth was stimulated, and the number of binucleate cells reduced, by addition to the culture of a bacterially expressed fusion protein containing the fibronectin-like SRYD motif and the zinc-binding (metalloprotease) domain of gp63. These observations support an additional role of gp63 in promastigote multiplication; the fibronectin-like properties of gp63 may be important in this process
Résumé
The genome of Leishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies of P-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). A P-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rniniexon- derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain of Leishmania donovani for coordinated genome mapping of this clinically important organism.
Résumé
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.
Résumé
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.
Résumé
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.
Résumé
The primary transcripts synthesized in vitro from a T3 DNA template by Escherichia coli RNA polymerase and by T3 phage-specific RNA polymerase have been characterized with regard to cleavage by RNase III and the size of the products of the cleavage reaction have been compared with those of in vivo T3 RNAs. It has been observed that the large RNA molecule synthesized in vitro by Escherichia coli RNA polymerase from the early region of T3 DNA are cleaved at specific sites by Escherichia coli RNase III to produce all the early mRNAs normally observed in T3-infected cells. In contrast, evidence presented here shows that some of the late T3 mRNAs are generated as direct products of transcription of late regions of T3 DNA by T3 RNA polymerase without mediation of RNase III, while many other late T3 mRNAs are formed by RNase III cleavage of two of the high molecular weight T3 RNA polymerase transcripts. These in vitro data appear to be in good agreement with the observed sizes of late T3 mRNAs formed in vivo in T3-infected RNase III-deficient and RNase III+ Escherichia coli cells.