title: Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major.
abstract: To complete its life cycle, protozoan parasites of the genus Leishmania undergo at least three major developmental transitions. However, previous efforts to identify genes showing stage regulated changes in transcript abundance have yielded relatively few. Here we used expression profiling to assess changes in transcript abundance in three stages: replicating promastigotes and infective non-replicating metacyclics, which occur in the sand fly vector, and in the amastigote stage residing with macrophage phagolysosomes in mammals. Microarrays were developed containing 11,484 PCR products that included a number of known genes and 10,464 random 1 kb genomic DNA fragments. Arrays were hybridized in triplicate and genes showing two-fold or greater changes in 2/3 experiments were scored as differentially expressed. Remarkably, only about one percent of the DNAs expression varied by this criteria, in either stage comparison. Northern blot analysis confirmed the predicted change in mRNA abundance for most of these (68%). This set of genes included most of those previously identified in the literature as differentially regulated as well as a number of novel genes. Notably, Leishmania maxicircle transcripts showed strong up-regulation in metacyclic and amastigote parasites, probably associated with changes in parasite energy metabolism. However, current data suggest that expression profiling using shotgun DNA libraries significantly underestimates the extent of regulated transcripts.
authors.affiliation: Department of Molecular Microbiology, Center for Infectious Disease Research, Washington University School of Medicine, Campus Box 8230, 660 South Euclid Avenue, Saint Louis, MO 63110, USA.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Akopyants
authors.forename: Robin S
authors.initials: RS
authors.lastname: Matlib
authors.forename: Elena N
authors.initials: EN
authors.lastname: Bukanova
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Smeds
authors.forename: Bernard H
authors.initials: BH
authors.lastname: Brownstein
authors.forename: Gary D
authors.initials: GD
authors.lastname: Stormo
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Beverley
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018503:Genome, Protozoan; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology; D008018:Life Cycle Stages; D051379:Mice; D008807:Mice, Inbred BALB C; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D015800:Protozoan Proteins