BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 significant protein level changes (Figure 5). These data suggest, firstly, that alteration of CLH levels in these cells proceeds via exclusively post-transcriptional mechanisms and, secondly, that despite clear perturbation to endocytic activity, this is not reflected in an obvious or coherent alteration to the underlying transcriptome. Significant transcriptome alterations between in vivo and in vitro cultured trypanosomes The vast majority of molecular studies on T. brucei are car- ried out using in vitro cultures. Recently, whole-genome microarray analysis indicated significant differences between in vivo and in vitro cultures of the malaria parasite Plasmodium falciparum [118]. Therefore, as an additional investigation into the influence of culturing conditions on intracellular transport we compared mRNA from blood- stream form MITat1.1 trypanosomes extracted from infected rats and from SMB cells cultured under standard conditions. A significant number of differentially abun- dant mRNAs are evident from the scattergram of these arrays (Figure 4L), and limma analysis indicated that 35 ORFs are significantly upregulated in the in vitro culture, while three ORFs are significantly downregulated (Table 3). These include ORFs for an RNA helicase, procyclin, CAP5.5, several small GTPases and their effectors and sev- eral kinases (Table 3). This expression pattern did not reveal an obvious functional pattern, in the sense that ORFs from many biological systems appeared affected. To eliminate the possibility that these alterations arise from cold-shock during isolation of MITat1.1 trypomastigotes, we also extracted RNA from SMB cultures incubated at 4°C for 1 hr. We observed very few changes between nor- mal and cold-shocked SMB cultures (Table 3), with only CAP5.5 and a Sec34-like ORF significantly upregulated in the cold treatment, both of which are procyclic markers. Given the rapidity of isolation of the mRNA from MITat1.1, which is achieved in less than one hour, and their incubation at 4°C during the procedure, which is expected to reduce metabolic activity, we consider these findings to be unlikely ascribable to factors associated with preparation of the samples as the differences in sev- eral mRNA levels are too great to have accumulated in such a short period. Therefore, these data highlight signif- icant differences between in vivo and in vitro cultured trypanosomes; this issue would be best pursued by whole genome microarray analysis which is beyond the scope of the present study. Discussion Coordinate control of gene expression is critical for life cycle progression in trypanosomes, and extensive remod- eling of the parasite cell is well documented. Here we used the intracellular trafficking system as a model for tran- scriptome changes. We selected this process on account of well documented alterations to endocytosis accompany- ing developmental progression, a good level of character- ization in trypanosomes, together with, by analogy to higher eukaryotes, the expectation that many gene prod- ucts will collaborate in regulation. Endomembrane trans- port involves ~10% of the protein coding potential within the eukaryotic genome [119,120] and position and time- dependent targeting, assembly of macromolecular com- plexes and cytoskeletal interactions all participate in con- trolling a system demanding high fidelity in delivery of molecules to specific subcellular destinations. Further, in higher eukaryotes, transcriptional flexibility and coordi- nation are associated with intracellular transport [17,121]. Due to the polycistronic mode of transcription in trypanosomes, changes in relative levels of mRNAs result primarily from alterations in the efficiency of nas- cent RNA processing or half-life [13]. Here we detected clear evidence for developmental alterations to the trypanosome transcriptome, but found very little flexibil- ity within a given life stage. Firstly, ~6% of transport-associated transcripts are devel- opmentally regulated, a greater fraction than reported for genome-wide transcription [14]. These data indicate that developmental remodeling of endomembrane transport is underpinned by alterations to mRNA abundance, as expected. We also observed prominent differential expres- sion of transport-associated mRNAs between trypano- somes cultured in vitro and those isolated from a mammalian host. A greater proportion of mRNAs were upregulated in the bloodstream stage compared to the insect form, correlating with increased endocytic activity. Prominent upregulated factors include Rab GTPases and SNAREs; significantly, simple upregulation of Rab and/or SNARE proteins is sufficient to augment specific transport pathways [116,122-124]. While the cohort of upregulated mammalian stage mRNAs is consistent with increased endocytic activity, there is evidence that the Rab5/Rab11 VSG endocytosis and recycling system [25,122,125] is only part of the developmental change. For PCFs, the upregulated cohort is consistent with lysosomal degrada- tive pathways playing a more prominent role [26]. Direct examination of several of the gene products implicated here is clearly required for a more detailed understanding of trypanosome differentiation. Secondly, and in clear contrast, extremely limited changes to mRNA levels were encountered in response to a wide variety of altered states, including major changes to cultur- ing conditions, insult with agents disrupting protein fold- ing and RNAi of critical mRNAs encoding factors essential for endocytosis and cell surface maintenance. Despite polycistronic transcription, a priori there is no compelling reason to assume that turnover of mRNA is uncoupled from signaling systems and, moreover, the observation that iron-deficiency leads to increased expression of the Page 18 of 28 (page number not for citation purposes)