A Physical Map of the Leishmania majorFriedlin Genome

Abstract

An extensive physical map of the Leishmania major Friedlin genome has been assembled by the combination of fingerprint analysis of a shuttle vector cosmid library and probe hybridization. The integrated data obtained for 9004 fingerprinted clones and 974 probes have placed 91.2% of the 33.58-Mb genome into contigs representing each of the 36 chromosomes. This first-generation map has already provided a suitable framework for both high-throughput DNA sequencing and functional studies of the L. major parasite.Leishmania is a flagellated protozoan parasite belonging to the family Trypanosomatidae. The 13 different species of Leishmania are responsible for a wide spectrum of human disease occurring mostly in the tropics and subtropics, although cases in North America have been documented (McHugh et al. 1996). Found in parts of Asia, the Mediterranean region, and South America, there are estimated to be over 2 million new cases of leishmaniasis each year in 88 countries, with 367 million people at risk (WHO 1995). Leishmaniasis thus constitutes a major threat to human health.The parasite is digenic; the extracellular, flagellated forms of Leishmania major differentiate from noninfective promastigotes to infective metacyclics in the alimentary tract of their dipteran (sandfly) vector. After inoculation into the mammalian host, the parasites become intracellular, entering macrophages and differentiating into nonmotile amastigotes. Infective parasites are able to resist the action of resident hydrolytic enzymes, inhibit activation of the oxidative burst, and mitigate the immunological attack of the host (Bard 1989;Killick-Kendrick 1990;Blackwell 1996;Mauel 1996). As a result, there is considerable worldwide interest in determining the factors that contribute to Leishmania's efficiency as a parasite.As a eukaryote, Leishmania is atypical. Genes are often organized in tandem repeats, many of which are transcribed polycistronically. Nonrepeated genes of related function can also occur in long transcription units, akin to operons in prokaryotes.Extensive post-transcriptional processing is then required to yield mature mRNAs, including the transsplicing of a 39-nucleotide spliced leader (SL, or miniexon-derived) RNA onto the 5Ј ends of all mRNA molecules (Miller et al. 1986;Ramamoorthy et al. 1996). In contrast, no introns have been detected, removing a requirement for cis splicing. Hence, Leishmania species are the subject of fundamental interest with respect to the evolution of gene regulatory mechanisms, as well as for the exploitation of these properties for the development of new methods of control.The application of standard genetic techniques to the study of Leishmania has been hampered by two factors: (1) No sexual cycle has been observed; and (2) the chromosomes do not condense at any phase of the cell cycle. However, the refinement of pulsed field gel electrophoresis (PFGE) methods over the last 10 years has enabled a molecular ''karyotype'' for the various Leishmania species to be obtained (Samaras and Spithill 19...

The Stage-regulated Expression of Leishmania mexicanaCPB Cysteine Proteases Is Mediated by an Intercistronic Sequence Element

“…This sequence element, however, does not appear to be present in the 3Ј-UTR of the L. mexicana CPB cDNA. 2 The results of this study, coupled with previous work that showed that the 3Ј-UTR and downstream intercistronic sequence of CPB2.8 can be used to generate high level expression of a reporter gene in amastigotes from constructs targeted to the rRNA locus (38), indicate that CPB2.8 mRNA also contains amastigote-specific stabilization sequences.…”

“…2 Comparative sequence analysis of the 3Ј-UTR of genes expressed at elevated levels in amastigotes revealed a 150-nucleotide region that is capable of inducing reporter gene expression specifically in amastigotes. This sequence element, however, does not appear to be present in the 3Ј-UTR of the L. mexicana CPB cDNA.…”

Identification and Characterization of T Cell-Stimulating Antigens from Leishmania by CD4 T Cell Expression Cloning

“…Cultures were pulsed with [ 3 H]thymidine for the last 18 h. Cultures were performed in triplicate and the figure is representative of three separate experiments. these Ags have been previously shown to induce T cell responses in leishmaniasis patients, although histone H2b, ␣-and ␤-tubulin, as well as malate dehydrogenase have been previously characterized in terms of biochemistry or molecular biology (27)(28)(29)(30).…”

Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites

“…The amplified fragment, nmt, was cloned into pGEM-T (Promega) and used to screen a gridded L. major Friedlin genomic cLHYG cosmid library (21). Of the 21 positive clones identified, 12 mapped to the same contiguous sequence on chromosome 33.…”