Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A-T rich genomes.
The advent of high-throughput methods for the analysis of global gene expression, together with the Malaria Genome Project open up new opportunities for furthering our understanding of the fundamental biology and virulence of the malaria parasite. Serial analysis of gene expression (SAGE) is particularly well suited for malarial systems, as the genomes of Plasmodium species remain to be fully annotated. By simultaneously and quantitatively analyzing mRNA transcript profiles from a given cell population, SAGE allows for the discovery of new genes. In this study, one reports the successful application of SAGE in Plasmodium falciparum, 3D7 strain parasites, from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated. It was demonstrated that P. falciparum is amenable to this technique, despite the remarkably high A-T content of its genome. SAGE tags as short as 10 nucleotides were sufficient to uniquely identify parasite transcripts from both nuclear and mitochondrial genomes. Moreover, the skewed A-T content of parasite sequence did not preclude the use of enzymes that are crucial for generating representative SAGE libraries. Finally, a few modifications to DNA extraction and cloning steps of the SAGE protocol proved useful for circumventing specific problems presented by A-T rich genomes.
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Harvard University, Building 1, Room 704, 665 Huntington Ave, Boston MA 02115, USA.
A
A
Munasinghe
S
S
Patankar
B P
BP
Cook
S L
SL
Madden
R K
RK
Martin
D E
DE
Kyle
A
A
Shoaibi
L M
LM
Cummings
D F
DF
Wirth
D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D020224:Expressed Sequence Tags; D015870:Gene Expression / Q000235:genetics*; D005821:Genetic Techniques; D018503:Genome, Protozoan; D015698:Genomic Library; D008928:Mitochondria / Q000235:genetics; D010963:Plasmodium falciparum / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D017421:Sequence Analysis
D018076:DNA, Complementary; D016054:DNA, Protozoan; D012333:RNA, Messenger