Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray.

TitleAnalysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray.
Publication TypeJournal Articles
Year of Publication2002
AuthorsDiehl S, Diehl F, El-Sayed NM, Clayton C, Hoheisel JD
JournalMol Biochem Parasitol
Volume123
Issue2
Pagination115-23
Date Published2002 Aug 28
ISSN0166-6851
KeywordsAnimals, Blotting, Northern, Escherichia coli, Gene expression, Gene Expression Profiling, Genes, Protozoan, HUMANS, Life Cycle Stages, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Transcription, Genetic, Trypanosoma brucei brucei
Abstract

A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.

Alternate JournalMol. Biochem. Parasitol.
PubMed ID12270627