TY - JOUR
T1 - Expression profiling of the Leishmania life cycle
T2 - cDNA arrays identify developmentally regulated genes present but not annotated in the genome
AU - Almeida, Renata
AU - Gilmartin, Brian J
AU - McCann, Sharon H
AU - Norrish, Alan
AU - Ivens, Alasdair C
AU - Lawson, Danial
AU - Levick, Mark P
AU - Smith, Deborah F
AU - Dyall, Sabrina D
AU - Vetrie, David
AU - Freeman, Tom C
AU - Coulson, Richard M
AU - Sampaio, Iracilda
AU - Schneider, Horacio
AU - Blackwell, Jenefer M
PY - 2004/7
Y1 - 2004/7
N2 - 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.
AB - 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.
KW - Animals
KW - Expressed Sequence Tags
KW - Gene Expression Profiling
KW - Gene Expression Regulation
KW - Leishmania
KW - Leishmania major
KW - Life Cycle Stages
KW - Molecular Sequence Data
KW - Oligonucleotide Array Sequence Analysis
KW - Protozoan Proteins
KW - Sequence Analysis, DNA
U2 - 10.1016/j.molbiopara.2004.03.004
DO - 10.1016/j.molbiopara.2004.03.004
M3 - Article
C2 - 15138070
SN - 0166-6851
VL - 136
SP - 87
EP - 100
JO - MOLECULAR AND BIOCHEMICAL PARASITOLOGY
JF - MOLECULAR AND BIOCHEMICAL PARASITOLOGY
IS - 1
ER -