title: Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome.
abstract: As genomic sequencing of Leishmania nears completion, functional analyses that provide a global genetic perspective on biological processes are important. Despite polycistronic transcription, RNA transcript abundance can be measured using microarrays. To provide a resource to evaluate cDNA arrays, we undertook 5' expressed sequence tag analysis of 2183 full-length randomly selected cDNAs from Leishmania major promastigote (days 3, 7, 10 of culture in vitro), and lesion-derived amastigote libraries. PCR-amplified inserts from 1830 of these cDNA representing 1001 unique genes were spotted onto microarrays, and compared internally with PCR-amplified open reading frames (ORFs) from 904 genes representing 842 unique genes annotated in the L. major genome. Microarrays were screened with RNA from procyclic, metacyclic and amastigote populations of L. major. Redundant clones on the array gave highly reproducible results, providing confidence in identification of stage-specific gene expression. Four hundred and thirty unique (i.e. non-redundant) stage-specific genes were identified. A higher percentage of stage-specific gene expression was observed in amastigotes ( approximately 35%) compared to metacyclics ( approximately 12%) for both cDNAs and ORFs, but cDNAs provided a richer source of regulated genes than currently annotated ORFs from the Leishmania genome. In mapping cDNAs onto the Leishmania genome, we noted that approximately 42% aligned to regions not recognised as genes using current predictive annotation tools. These genes are highly represented in our stage-specific genes, and therefore represent important drug targets and vaccine candidates. Careful annotation of cDNAs onto the Leishmania genome will be important before producing the next generation of oligonucleotide arrays based on annotated genes of the genomic sequencing project.
authors.affiliation: Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK.
authors.forename: Renata
authors.initials: R
authors.lastname: Almeida
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Gilmartin
authors.forename: Sharon H
authors.initials: SH
authors.lastname: McCann
authors.forename: Alan
authors.initials: A
authors.lastname: Norrish
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Ivens
authors.forename: Danial
authors.initials: D
authors.lastname: Lawson
authors.forename: Mark P
authors.initials: MP
authors.lastname: Levick
authors.forename: Deborah F
authors.initials: DF
authors.lastname: Smith
authors.forename: Sabrina D
authors.initials: SD
authors.lastname: Dyall
authors.forename: David
authors.initials: D
authors.lastname: Vetrie
authors.forename: Tom C
authors.initials: TC
authors.lastname: Freeman
authors.forename: Richard M
authors.initials: RM
authors.lastname: Coulson
authors.forename: Iracilda
authors.initials: I
authors.lastname: Sampaio
authors.forename: Horacio
authors.initials: H
authors.lastname: Schneider
authors.forename: Jenefer M
authors.initials: JM
authors.lastname: Blackwell
mesh_terms: D000818:Animals; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007891:Leishmania / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D015800:Protozoan Proteins