A significant drop in cam promoter activity has previously been described by Crabb et al. (1996), to occur upon deletion of 87 bp of the intergenic sequence between nucleotides 625 bp and 537 bp upstream of the cam ORF. To investigate these findings in more detail, and the possible function of the downstream sequence, a range of deletion mutants was selected for analysis by transfection.
3.5.2 RESULTS 1: Deletion mutant library
A culture at a parasitaemia of 10% was transfected with approximately 100 pg of each pEXO construct, as well as the control vectors pHCl-CAT and pHEP.luc. DNA for transfection was carefully quantified and diluted into 1 x TE pH 8.0 to a final concentration of 3.3 pg/pl, and 30 pi (100 pg) used for each electroporation. At 22 hr and at 46 h post transfection, samples were harvested, and split into two, to allow the reporter assays to be performed in duplicate. The transfected parasites were
the ratio of CAT activity to luciferase activity was calculated and plotted against 5' UTR length (Figure 3.18 and included in Figure 3.19).
The data generated in this experiment showed a 50% drop in cam promoter activity on removal of the sequence between 625 and 539 bp upstream of the cam
start ATG. Activity dropped to 20% that of the full-length promoter on removal of a further 130 bp (pEXO.410) and constructs containing 313 and 270 bp were found to contain 3 and 1.6% of promoter activity respectively. These findings are in general agreement with those published by Crabb et al. (1996). However, Crabb et al. (1996) describe an 80% drop in activity on removal of the sequence between 625 and 537 bp upstream of the start ATG and residual activity of approximately 5 or 6% for the construct containing 354 and 278 bp of the promoter.
The luciferase activity detected for each construct varied substantially (Figure 3.17A). The luciferase expression cassette in each of the constructs studied was identical and each transfection was performed with equal amounts of DNA, as measured by spectroscopic analysis (section 2.1.6). Variations in transfection efficiency had not been expected to be so great and therefore the amount of DNA in samples used to perform the transfection was measured. The DNA preparations turned out to be at a range of concentrations, from 1 tig/pl for the control vector pCaRp33L, to 4 pg/p.1 for pEXO.119, i.e. each electroporation had been performed with a different quantity of DNA. The reasons for this discrepancy are unclear but at the high concentration needed for transfection (3.3 pg/pl) the DNA solution becomes viscous and dividing it into aliquots is problematic. In later experiments the DNA was prepared as described in section 2.1.6.
Chapter 3
3.5.3 RESULTS 2: Detailed analysis of the region driving maximal gene expression
To investigate the area of the intergenic region between 625 and 400 bp which, in the previous experiment had been shown to possess 80% of the promoter activity, a range of deletion mutants covering this area, differing in length by approximately 20 bp, was selected for analysis by transient transfection. A culture at a parasitaemia of 10% was transfected in duplicate with 100 pg of each construct, each harvested sample of parasite material was divided into two and the appropriate assay performed in duplicate.
The previously detected 80% drop in activity on removal of 225 bp of promoter sequence was still observed, and no activity above background was seen with construct pEXO.232 or pEXO.l 19 (data not shown). However, the results from the duplicate transfections using constructs ranging from 597 bp to 539 bp in length were contradictory, and it was not possible to obtain a more detailed map of putative control elements than that previously described. The experiment was repeated using fewer constructs to limit the error introduced by handling so many samples and a consistent profile of activity was obtained. Between 600 bp and 500 bp a 50% drop in activity was detected. A fiirther 30% drop in activity was then seen between 500 bp and 400 bp, and no activity above background was seen in constructs pEXO.232, pEXO.l 19 or pEXO.9 (data not shown). Again, significant differences in activity were seen between duplicate transfections for the constructs containing small differences in the length of the promoter region. The experiment was performed once more, extreme care being taken to ensure that all the samples were exposed to the
before each assay. However, once again a large variation in reporter activity was detected between closely related constructs (data included in Figure 3.19) and the relevance of the findings was hard to interpret. Since the increases and decreases in activity detected over the region were not reproducible i.e. construct X would display less or more activity than neighbouring constructs in different experiments, it was apparent that the sensitivity of the assay was limiting when looking at small changes in activity. To build a more thorough profile of activity than that initially obtained (section 3.5.1) would involve repeating the experiments several times and using a few constructs at a time to limit handling. This would be a very lengthy process, and at this point was considered unlikely to shed much light on the question at hand i.e. which region of the cam S’ UTR harbours control elements i.e. TF binding motifs. A combination of transfection inefficiency and the error margins of the assays led me to believe that I was not going to be able to identify small regions of significant activity using this method, nor eventually study the promoter activity using point mutation studies as was originally hoped.
3.5.4 RESULTS 3: Identification of a minimal promoter element
Though detection of significant and reproducible differences in activity proved difficult, it was possible to address the question of minimal promoter activity. To look at this, several of the shorter constructs, pEXO.437, pEXO.420, pEXO.358, pEXO.313, pEXO.270, pEXO.168, pEXO.l 19 and pEXO.9, were selected for transfection and pHEP.luc and pHCl-CAT were included as controls. To increase the sensitivity of the assay a 20% parasitaemia culture was transfected with 100 pg of
Chapter 3 analysis. Luciferase activity was detected with each construct and used to normalise CAT activity as previously described. CAT activity above background was detected in parasite material derived from cultures transfected with pEXO.437, pEXO.420, pEXO.358, pEXO.313, pEXO.270 and pEXO.232 and no activity was detected with parasites transfected with pEX0168, pEXO.l 19 or pEXO.9. Promoter activity, demonstrated by normalised CAT activity, decreased rapidly over the first 3 constructs, dropping from approximately 40% of maximal promoter activity to approximately 5% (313 and 270) and only 1% of maximal activity was detected with pEXO.232 (data included in Figure 3.19). The data suggest that a minimal promoter lies between 350 and 250 bp upstream of the cam start ATG.