confidence of the feasibility of the comparison between the two techniques and follows the central dogma of Gene-Transcription-Translation. However, perhaps
148 more significant finding was the discrepancies between proteomic data and transcriptomic data.
The presence of proteomic evidence in the absence of detectable transcriptomic evidence has been noted in several other studies [235-237], as well as in Plasmodium, Cryptosporidium and Neospora as shown in this chapter. The detection of these genes by proteomic studies reflected the high sensitivity of the proteomic approaches. In the case of T. gondii, by combining expression evidence from microarray, EST and SAGE, there were 6736 release 4 genes which have transcriptomic evidence, representing 86.4% of the entire genome. It is unlikely that all those 6736 genes are expressed in the tachyzoite stage, especially with the inclusion of 75% of all the genes assayed on microarray; it is likely to include genes expressed in other life stages. Considering the vast coverage of transcriptomic data, the identification of the 60 T. gondii genes for which no transcripts were observed is more fascinating. One possibility of the cause of discrepancies between proteomic data and transcriptomic data is technical limitations, whereby the same level of analytic resolution is hard to reach between the two techniques. Other possibilities involve biological explanations such as selective protein degradation and variations in protein turn-over rates [240, 241] as well as post-translational regulations such as mRNA decay and translational repression [242-244].
Three examples of those 60 genes that have exclusive proteomic evidence indicated in section 5.3.2 were „tubulin beta chain, putative‟ (28.m00301, 128 peptide hits), „thioredoxin, putative‟ (42.m03331, 57 peptide hits) and „coatomer protein gamma 2-subunit, putative‟ (59.m00090, 53 peptide hits). These three genes were identified by large numbers of peptide identifications in the absence of transcript evidence in
149 tachyzoite stage. Interestingly, although no evidence have been found from Day 6 and RH libraries of SAGE experiment, both the tubulin beta chain, a component of microtubule that is critically important for shape and apical polarity [357, 358] and thioredoxin, a vital component in the antioxidant system of T. gondii, which is essential for the adaption and survival of the parasites in macrophages and other immune effecter cells [359] were detected in an earlier library-Day 4 (a VEG primary library representing a mixture of sporozoites and early stage of tachyzoites gene expression). It is possible that sufficient mRNA was produced in the early stage of tachyzoites to retain the required level of protein expression whereby no further mRNA is required in the later stage of the development.
This observation coincides with the finding of the SAGE study that a major shift in gene expression happens from Day 4 to Day 6 libraries [272]. In fact, another 11 genes out of those 60 genes have been detected in the Day 4 SAGE library, including a cell cycle control protein, putative (641.m01576, 7 peptide hits). The major shift in gene expression patterns could also partly explain the smaller size of the SAGE dataset and the reduced overlap with proteomic data compared to EST and microarray data, as shown in Figure 5.2. The SAGE data provided a more accurate measurement of mRNA expression at the specific Day 6 time point (and closely correlated RH strain [272]), while EST data were collected from a larger collection of various time points within the tachyzoite stage, and the arbitrary 25% cut-off used for microarray dataset is likely to include and exclude some genes that are not expressed at this life stage, as discussed earlier.
Interestingly, while the SAGE study indicated the closest correlated mRNA expression to the laboratory strain RH library was the Day 6 library, the proteomic data actually had a larger overlap with an earlier Day 4 SAGE library. In total, 762
150 genes detected by proteomic data were shared with Day 4 SAGE library while only 428 genes were shared with Day 6 SAGE library. The finding that the expression profile of proteomic data is closer to an earlier time point of transcriptomic data is likely to reflect the rapid changes in gene expression profiles and the temporal differences between mRNA and protein levels.
While the discrepancies between transcriptome and proteome have been observed in T. gondii and other Apicomplexan parasites in this study, the analysis that was designed to check for common features of proteins with low levels of transcription across Apicomplexa have only identified a few candidates at the level of orthology. The larger overlap of orthologues observed between T. gondii and N. caninum than the overlaps with P. falciparum in all three comparisons shown in Figure 5.4 is likely to reflect a closer phylogenetic distance between T. gondii and N. caninum, although no specific class of proteins can be highlighted. In all the three Apicomplexan parasites analysed, there was no apparent underlying rule that can explain the discrepancies between proteomes and transcriptomes.
There were some interesting candidates in the comparison, such as a coatomer protein gamma 2-subunit, putative, 59.m00090; ToxoDB annotation. It has been shown in T. gondii that although no transcript evidence has been found with microarray, EST and the three libraries of SAGE, convincing peptide evidence (53 peptide hits) were detected. The protein orthologues of this gene have been consistently detected in P. falciparum, N. caninum and C. parvum but no EST evidence has been found in N. caninum and C. parvum, and only a single corresponding EST has been seen in a P. falciparum blood-stage EST library. Coatomer protein gamma 2-subunit resides in Golgi-derived vesicles which mediate both selective and non-selective transport between the ER and Golgi and/or within
151 the Golgi cisternae [360, 361]. It is possible that this protein has a very long turn- over rate or an extremely high copy number from a single transcript. The precise reason of the consistent discrepancies observed between proteome and transcriptome of this gene across several Apicomplexan parasites and various life stages is very interesting and requires further investigation.