BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 with a possible role in membrane transport are differen- tially expressed in T. brucei; 64 upregulated in BSF, 37 in PCF, i.e. 8% and 5% respectively. 25% of the most significantly developmentally regulated genes were validated by qRT-PCR analysis, using the same mRNA samples as used for the microarray analysis. The vast majority of ORFs tested exhibited congruence between qRT-PCR and microarray analysis, i.e. differential regulation in the same direction (BSF or PCF, Table 1). Significantly, all ORFs with a ratio above 2.5 on the array also gave a significant ratio (i.e. above 2) in qRT-PCR. Two genes with known developmental regulation, the clathrin heavy chain [10,16] and TbMSP-A [52], did not demon- strate significant differential regulation by microarray analysis, but highly significant ratios were obtained by qRT-PCR (data not shown). Various factors may be responsible for this array artefact, which were not pursued further; however these data specifically highlight exam- ples of false-negative data in microarray analysis. Differential expression of trafficking factors in trypanosomes Considering the differentially expressed genes by func- tional class indicates that a broad remodelling accompa- nies differentiation, i.e. developmental changes are not restricted to specific protein classes or pathways. Specifi- cally, members of the chaperones, some proteases and non-PI kinases are upregulated in PCF, while the small GTPases, additional proteases, SNARE proteins and PI kinases represent the major factors upregulated in BSFs (Figure 2, Additional file 6). An increased requirement for chaperones may be due to the greater heterogeneity of the procyclic cell surface compared to the bloodstream form, the latter being dominated by VSG. A differential require- ment for secretory pathway proteases is consistent with distinct roles for these molecules between the two life stages, while upregulation of both small GTPases and SNARE proteins in BSFs is broadly consistent with increased activity within the trafficking system in the mammalian stage. Specificity and control factors The most abundantly expressed small GTPases at the mRNA level are Ran, ARL3A, both Rab1A and Rab1B, and several members of the Arf subfamily (Figure 3). Ran is a very abundant protein in most cells, while high expres- sion of Arf1, Rab1, and ARL3A possibly suggest a consid- erable emphasis on flux through the ER and Golgi complex and flagellum biogenesis respectively [53-55]. Developmentally expressed small GTPases are restricted to the BSF. This cohort includes markers for the recycling endosome, Rab11 [15], the endosomes/Golgi, TbArf1 [53], and the flagellum, TbArl3A [55]. Identification of Rab11 is consistent with earlier work [15] and our data 100% n itoa 80% t sen 60% e r rep 40% t n rce 20% e P 0% Increased in BSF Increased in PCF Equivalent Functional category gSFriigognuuifpriceeadn2btly fduenvcetliopnmalecnlatsaslly expressed trypanosome genes Significantly developmentally expressed trypano- some genes grouped by functional class. Genes repre- sented by array oligonucleotides were grouped by function, based on sequence similarity to annotated sequences, domain annotation, GO terms and additional criteria. The proportion in each functional class significantly upregulated in BSF are shown in red and in PCF in green. The remaining fraction of genes in each class are shown in grey. The first bar illustrates the proportion of developmentally regulated tran- scripts in the entire studied gene cohort. For raw data [see Additional file 6]. provide the first evidence for developmental regulation of TbArf1 and TbArl3A. The data also identify Rab1B, RabX3, Ras-like GTPases TbRX3 and TbRHP, and a putative GTPase with an Hsr1 domain (Tb927.4.2380) as develop- mentally regulated; with no experimental information for these proteins the significance of these changes is not clear [29,56]. Importantly, we see no strong developmental reg- ulation for any of the putative GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) included in the study. Some developmental regulation of TBC family Rab-GAPs is detectable by qRT-PCR, but at lev- els unlikely to be detected by the array (C. Gabernet-Cas- tello and MCF, unpublished data). In addition to a number of small GTPases, including Rab subfamily members, five predicted SNARE mRNAs exhibit increased expression in BSFs (Table 1). While no direct experimental information is available for these molecules, Tb09.211.3920, the most abundant Qa SNARE at the mRNA level (Figure 3), is likely involved in post-Golgi transport on account of similarity to trans-Golgi network (TGN) SNAREs SYP42 from Arabidopsis thaliana and syntaxin16/Tlg2p from S. cerevisiae [57,58], while the L. major orthologue, LmjF35.2720, localises close to the Golgi complex [59]. Tb10.70.7410 and Tb09.160.2420 are similar to the A. thaliana early endosomal SNARE VAMP727 and the ER-located SYP72, respectively [58], Page 8 of 28 (page number not for citation purposes)