BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 Flexible le1 v e l A N R2 m e v i t a l 3 e R Inflexible lve1 e l A N R2 m e v i t le3 a R Mammal Mammal Differentiation signal (temperature) Differentiation signal (temperature) Insect Insect TFirgaunsrceri6ptional flexibility and inflexibility in differentiation and responsiveness Transcriptional flexibility and inflexibility in differentiation and responsiveness. Upper panel: flexible system. Gene cohorts 1 and 3 are developmentally regulated, and either highly expressed or not expressed; examples of these types of gene products are the trypanosome surface antigens, VSG in the bloodstream form (red) and procyclin in the insect stage (green). The vast majority of genes fall into cohort 2, where, for example, either small or large changes to transcription could result from alterations to the environment (light and dark blue), or a more continually altering transcriptional profile is present that may seek to track changing conditions (purple). This behavior may propagate from one life stage to the next (light and dark blue) or be lost (purple) resulting in altered transcriptional flexibility for genes between life stages. Such a profile is found in higher eukaryotes, including humans and yeast, and probably also many protists, including E. gracilis. Lower panel: inflexible sys- tem. In this model gene cohorts 1 and 3 behave as before, but transcription of the genes in cohort 2 remains unchanged. The relative levels of mRNAs from the genes in this cohort may remain constant following differentiation (light blue) or be signifi- cantly altered (dark blue and purple). Such a profile is observed here for T. brucei and has been reported previously for P. falci- parum, and is potentially a result of a parasitic life style where the host is responsible for provision of a homeostatic environment. including major changes to culturing conditions, insult with agents disrupting protein folding, and RNAi of criti- cal mRNAs encoding factors essential for endocytosis and cell surface maintenance, elicited extremely limited changes to the transcriptome. Besides alterations in expression of the transferrin receptor upon iron stress, which is transcribed by Pol I, the vast majority of trypano- some chromosomal Pol II-derived mRNAs are unmodu- lated. These findings imply a very limited capacity of trypanosomes to respond to altered circumstances in vivo, except via pre-determined differentiation pathways (Fig- ure 6). Hence mRNA levels are insensitive to external stimuli and most responses are mediated at the proteome level. These data suggest a new view of the trypanosome life cycle as a progression through sequential, inflexible transcriptional profiles that serve to adapt the parasite to each new host environment. As similar transcriptional rigidity is suggested for the malaria parasite, Plasmodium falciparum, but transcriptional flexibility is found in the closely related and free-living protist, Euglena gracilis, it is likely that loss of transcriptional flexibility arose through the parasitic lifestyle. Methods Open reading frame selection We selected ORFs encoding components of the trypano- some transport system using the geneDB interface [137]. The genome was parsed with four criteria; (i) annotation keywords, including description information and wild- cards, for protein classes involved in endocytosis and membrane trafficking (e.g. clathrin, adaptin, SNARE, Rab, ADP-ribosylation factor, coatomer, Sec, Vps), (ii) Pfam/ Interpro domain annotation (e.g. ARF/SAR superfamily, C2, ENTH, PH, SH3, SNF7, VHS, BAR), (iii) "Biological Process" Gene Ontology (GO) annotation (e.g. endocyto- sis, vesicle mediated transport, GTPase activity) and (iv) sequence similarity by BLAST. The target gene selection was augmented by parsing the literature for candidate fac- tors, i.e. yeast and mammalian proteins that have been functionally demonstrated to be involved in trafficking. We thus identified 673 ORFs for inclusion. Array construction Oligonucleotides were designed with OligoArray 2.0 [138] specifying a 67 mer within one kilobase of the 3'- Page 21 of 28 (page number not for citation purposes)