The transcriptomes of Trypanosoma brucei Lister 427 and TREU927 bloodstream and procyclic trypomastigotes

Abstract

We describe developmentally regulated genes in two strains of Trypanosoma brucei: the monomorphic strain Lister 427 and the pleomorphic strain TREU927. Expression patterns were obtained using an array of 24,567 genomic fragments. Probes were prepared from bloodstream-form or procyclic-form trypanosomes. Fourteen procyclic-specific and 77 bloodstream-specific signals were obtained from sequences matching variant surface glycoprotein or associated genes, and a further 17 regulated sequences were repetitive or transposable-element-related. Two hundred and eighty-six regulated spots corresponded to mRNAs from other protein-coding genes; these spots represent 191 different proteins. Regulation of 113 different genes (79 from procyclic forms, 34 from bloodstream-forms) was supported by at least two independent experiments or criteria; of these, about 60 were novel. Only two genes -encoding HSP83 and an importin-related protein -appeared to be regulated in the TREU927 strain only. Our results confirmed previous estimates that 2% of trypanosome genes show developmental regulation at the mRNA level.

Functional analysis of Trypanosoma brucei PUF1

“…RNA preparation and analysis methods were similar to those previously described [27,28]. Briefly, for the genome-wide analysis of gene expression, 15 g of total RNA was incubated for 5 min at 65 • C with 500 ng of oligo d(T) [12][13][14][15][16][17][18] (Amersham), 30 mM dAGT-mix, 2 mM dCTP and 2 mM Cy3 (or Cy5) labelled dCTP (Amersham).…”

“…Arrays of 23,000 [27] or 24,000 [28] random 2 kb genomic fragments on glass slides were pre-incubated in 5× SSC, 0.1% SDS and 1% BSA for 1 h at 42 • C, dipped in water then isopropanol and allowed to dry. Hybridization with mixed Cy3-and Cy5-labelled cDNAs was in 50% deionised formamide, 5% dextran sulfate, 3× SSC, 1% SDS and 5× Denhardts solution at 42 • C for 16 h, using 24-60 mm cover slips and glass array hybridisation cassettes (Ambion).…”

“…Slides were then washed for 5 min each in 1× SSC, 0.2% SDS, 0.1× SSC, 0.2% SDS, and 0.1× SSC at room temperature. The results were analysed as previously described [27,28] using M-CHIPS software [29,30]. To choose regulated clones for sequence analysis we selected only those with the following parameters-fitted intensities: at least 50,000; min/max-separation: 0.2; ratio for RNAi induced versus uninduced: at least 2.0 or −2.0.…”

“…To find out if perturbation of PUF levels in T. brucei influenced steady-state levels of specific mRNAs, we used microarrays to look for mRNAs which were changed at least two-fold after PUF1 RNAi or over-expression [28]. For both procyclic and bloodstream-form RNAi cell lines (the procyclic knockout was obtained later), the down-regulation of PUF1 was readily detected but no other effects on gene expression were found.…”

“…Extracts from cells expressing the TAP tag alone served as controls for cDNA incorporating the alternative fluorescent label. The two labelled preparations were mixed, hybridised to genomic microarrays [28], and spots which hybridised preferentially to the sample from the PUF1-TAP preparations were identified and the inserts sequenced. The table lists the identities of spots which gave >3-fold more fluorescence (min/max separation filter 0.1) from the PUF1-TAP cDNA than from the TAP-only cDNA in each of two independent purifications.…”