From the genome to the biology of Leishmania: a comprehensive approach

Abstract

The health of information generated by large-scale genome sequencing unveils the frenzy to come in biological and medical research. The Leishmania genome project, now focused on sequencing, starts to uncover structural information on genome organization and the very first attempts to conduct functional analysis of generated data are in its early steps. In that direction it is important to note that the peculiar genetic traits of this organism might play an important role in defining strategies and paths to follow. The relevance of biological information to be gathered lies at the center of the matter. In this spirit we bring the proposal presented here. The aims of our proposal are divided in six theme-related projects. In the first one we aim to generate the complete sequence for L.major chromosome 2, as part of the international consortium involved in the Leishmania genome project. The basis of the sequencing project will rely on the minimum file path of fingerprinted chromosome 2 commits and in a chromosome specific shotgun library. We will also be using the mariner in vitro transposition system as a support strategy for sequencing completion. These transposable elements are the basis for the definition of a systematic knock out strategy and gene trapping of transcribed sequences within this chromosome. Other two projects are centered on understanding structural sequences known to be functionally relevant. In this regard the study of centromeric sequences is important to understand the phenomenon of chromosome segregation in Leishmania. We have tackled that matter through the analysis of maintenance dynamics of linear recombinant vectors transected into the parasite. We have already identified clones presenting altered maintenance kinetics in the parasite that might carry sequences relevant to the stability of linear epitomes. We will further dissect such region confirming its centromere function and evaluating its distribution within the genome. Telomere regions are also objects of interest. We are currently characterizing three genomic clones carrying three distinct telomere regions. Transfectants over expressing each one of these recombinants were used to define a transcriptional map and the presence of repetitive sequences within the region. Our aim is to refine this characterization by sequencing some of the most extreme transcripts. We will also further characterize a telomere clone known to impair promastigote growth in vitro. This project brought to our attention the importance of the position or direction of a transcript within the episome to the level of the induction of expression. To address the issue we will use DHFRTS null mutants complemented with episomes carrying the DHFRTS gene in various position and direction in relation to the selectable marker present in the vector backbone. Physically mapping of chromosome 2 revealed a genomic clone bearing the miniexon tandem array which was used as tool to demonstrate that the over expression of this gene leads to a substantial attenuation of virulent strains of Leishmania. These mutants will now be tested as live vaccines against leishmaniases and will also be used to shed light on the poorly understood virulence/attenuation phenomena. The last project deals with the potential presence of selenoproteins in Leishmania. Our interest in this matter was raised by our studies of the abundant class of genes of unknown function identified within the leishmania genome project. (AU)