A survey of the Leishmania major Friedlin strain V1 genome by shotgun sequencing: a resource for DNA microarrays and expression profiling

Abstract

Leishmania are pathogenic trypanosomatid protozoans responsible for a diverse spectrum of human diseases. These parasites exhibit a dimorphic life cycle, consisting of extracellular promastigotes that reside within the midgut of the sand fly vector, and intracellular amastigotes that reside within the phagolysosome of host macrophages. How Leishmania differentiate, survive and multiply in such inhospitable environments is a challenging question relevant to disease control. In the last 10 years, genetic tools have been developed which permit a variety of modifications to the parasite genome, using expression vectors and gene knockout technology [1]. A genome project has been initiated, and the sequence of several chromosomes has recently been completed [Refs. [2] [3] [4]; and http:// www.ebi.ac.uk/parasites/leish.html]. The combination of extensive genome sequence information and reverse genetic tools permits powerful new approaches for understanding gene function in this parasite. Experimenters must now confront the question of how best to choose amongst the 10 000 proteins encoded by the parasite genome for further study. This challenge is compounded by the fact that most Leishmania genes show no obvious sequence similarity to those encoding any known proteins [Ref. 4, this work]. One criterion for prioritizing genes for further study is based on the concept of expression profiling, in which the level of mRNA abundance is determined for thousands of genes simultaneously [5]. This may be accomplished by hybridization of total labeled mRNA (or cDNA) preparations to arrays of DNAs on glass slides, followed by quantitation of specific hybridization by fluorescence [6,7]. Comparative analysis of hybridizations using different preparations permits, for example, the identification of genes that are expressed in a stagespecific manner. Although many routes of gene regulation do not involve changes in mRNA abundance (a key point in Leishmania and trypanosomatid parasites, given their reliance upon polycistronic transcriptional mechanisms [8,9]) a number of mRNAs implicated in Leishmania virulence have been found to exhibit differential regulation [8,10–12]. DNA microarrays typically exploit well-characterized collections of cDNAs, or completed genome sequences from which oligonucleotide probes can be synthesized directly on a chip [5]. As neither of these resources is yet available for Leishmania, we elected to pursue an alternative approach based upon random shotgun DNA sequences. This approach is feasible in trypanosomatid parasites due to their high gene density Note : The sequences reported in this work have been deposited in the GSS section of GenBank. * Corresponding author. Tel.:+1-314-7472630; fax: +1-3147472634. E-mail address: beverley@borcim.wustl.edu (S.M. Beverley).