Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray.
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.
Diehl
Susanne
S
Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, 69120 Heidelberg, Germany.
Diehl
Frank
F
El-Sayed
Najib M
NM
Clayton
Christine
C
Hoheisel
Jörg D
JD
D000818:Animals; D015152:Blotting, Northern; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D017125:Genes, Protozoan; D006801:Humans; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000302:isolation & purification