BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 [18], and a putative UTP-glucose-1-phosphate uridylyl- transferase 2 (Tb10.389.0330) is related to human UGP2, required for clathrin-mediated endocytosis [18]. A glyco- somal glycerol kinase (Tb09.211.3540) related to human CARKL, which is required for caveolin/lipid raft-mediated endocytosis [18], was upregulated in PCF, although equivalent levels of specific activity for this enzyme have been reported for both life stages [80,87]. An arginine kinase (Tb09.160.4560) involved in the management of cellular ATP energy reserves [88] is similar to human CKB, required for clathrin-mediated endocytosis [18]. A puta- tive dephospho-CoA kinase (Tb927.6.710) which cata- lyzes the final step in CoA biosynthesis, is related to human DPCK, which is required for caveolin/lipid raft- mediated endocytosis [18]. A phosphoglycerate kinase (Tb927.1.720), similar to human PGK1, which is involved in both clathrin-mediated and caveolin/lipid raft-mediated endocytosis [18] was up in PCF, consistent with previous analyses for T. brucei PGKB [89], although the oligonucleotide used does not allow us to differentiate PGK isoforms. A putative Ser/Thr and Tyr dual specificity phosphatase (Tb10.406.0240) possibly involved in MAP kinase signalling, is also upregulated in PCF. Taken together these data suggest multiple kinases are involved in control of trafficking in both life stages, with more sig- nalling kinases upregulated in BSF, and these data provide a number of candidates for further analysis. Ubiquitylation Covalent attachment of ubiquitin to surface molecules is an important mechanism for internalization and lyso- somal targeting [90,91], and is present in trypanosomes [92,93]. Five ORFs likely to participate in the trypano- some ubiquitylation system are upregulated in BSF; Tb09.211.3610, an ubiquitin-activating E1 enzyme, Tb927.4.2710, an ubiquitin-conjugating E2 enzyme, Tb927.6.2370, an ubiqutin ligase E3, Tb11.46.0014, an ubiquitin carboxyl-terminal hydrolase similar to S. cerevi- siae Doa4p protein, and Tb10.70.1130, which contains two putative ubiquitin interaction motifs, but otherwise has no predicted function. Upregulation of a near com- plete pathway for protein ubiquitylation and deubiquit- ylation in BSFs is suggestive of stage-specific ubiquitylation, but clearly direct evidence is required [94]. RNAi of the E1 Tb09.211.3610 shows a strong growth defect but does not affect the stability of ISG65, a surface protein subject to ubiquitylation [93]. A protein contain- ing a VHS domain (Tb11.01.2540) which is normally found in the Vps27 component of ESCRT 0, and a putative Vps46 component of the ESCRT III-associated complex (Tb11.02.0580) are upregulated in procyclics, further sup- porting differential regulation of the ubiquitin sorting sys- tem. Hypothetical open reading frames A considerable fraction of the analysed ORFs are anno- tated as hypothetical, and were included on account of sequence features shared with characterized trafficking factors. Several of these exhibit developmental regulation. For example, the C2 domain is a Ca2+-dependent mem- brane-targeting module [95-97]; two ORFs with predicted C2 domains, Tb09.160.3240 and Tb927.7.3550, are upregulated in procyclics, and one, Tb927.4.2080, is upregulated in BSF. Interestingly, Tb927.7.3550 localises between the inner face of the plasma membrane and the sub-pellicular corset of microtubules in T. brucei, and includes a highly charged region characteristic of a tubu- lin-binding domain [98]. RNAi evidence indicates a clear role for Tb927.7.3550 in cytoskeletal function [98]. Other hypothetical genes that show significant differential regulation at the mRNA level are, in PCF: Tb927.7.2640, which contains part of a Sec34 domain (although it is not a true COG3/Sec34 orthologue [41]), and Tb11.47.0022, which contains a signal peptide and a trans-membrane helix; both ORFs are highly conserved between trypano- somatids but have no identifiable orthologues in other organisms. In BSF: Tb11.01.4460, which contains a cysteine-rich repeat region found in the low-density lipo- protein (LDL) receptor, and Tb927.5.4520, an extended coiled-coil protein are upregulated. The flagellum adhe- sion glycoprotein fla1 was upregulated in PCF, as has been reported previously [99], but the closely related fla2 [100] was upregulated in BSF, indicating that the two gene products may have similar functions but distinct expres- sion patterns. Surface antigens Messages for BSF-specific ESAG6/7 and ISG65/75 surface antigens are highly abundant (Figure 3). By contrast, CRAM, a possible lipoprotein receptor abundant in PCF, and BARP, a putative glycolipid-lipid raft associated pro- tein are not so highly expressed (Figure 3). Interestingly, some of the oligonucleotides for BARP showed upregula- tion in PCF and some in BSF (Table 1); BSF-specific and epimastigote-specific BARP expression have been reported previously [12,44]. The differential expression levels for the ISG75 mRNAs (Figure 3) represent mostly the degen- eracy of each oligonucleotide [see Additional file 2], but the mRNAs for the ISG65 genes on chromosome 5 (Tb927.5.1390-1410-1430) are clearly more abundant than those for the ISG65 genes on chromosome 2 (Tb927.2.3280-3290-3300-3310). We also included oli- gonucleotides for several ISG-like proteins, four of which, Tb927.5.310, Tb927.5.420, Tb927.5.430, and Tb927.5.630, are significantly upregulated in BSF, sug- gesting additional and so far uncharacterised develop- mental modification of the parasite surface. Page 12 of 28 (page number not for citation purposes)