Criterios de selección.

The effects of the deletions on the 625 bp cam promoter activity were analysed by quantifying their ability to drive expression of CAT enzyme in transiently transfected P. falciparum cultures. To allow quantitative analysis of the changes in activity between individual transfections and different experiments, CAT activity was to be normalised against the luciferase activity driven by the intact hrp

in

region on the same construct. To enable a valid comparison to be made between enzyme activity and promoter activity it was first important to define assay conditions for both reporter gene products that gave a linear relationship between enzyme concentration and enzyme activity. It was also necessary to show that, for any given construct, the relationship between the level of the two reporter enzymes remained constant over a range of input DNA levels and that the two reporter cassettes were truly independently regulated. A linear relationship between enzyme concentration

enzymes. The effect of transfection efficiency on the ratio between the reporter activities was addressed by using varying amounts of input DNA to simulate varying efficiency of transfection.

3.3.1 The luciferase reporter gene and assay

Commercial (Promega) luciferase is derived from a common North American firefly,

Photinus pyralis. The gene product catalyses the breakdown of luciferin to produce

light in a two step reaction:

(I) Luciferase + Luciferin + ATP --- ► Luciferase-Luciferin-AMP +PPi

(II) Luciferase-Luciferin-AMP + 0% ► Luciferase + Oxyluciferin + AMP+ CO2 + Luminescence

(d eW E T 1 9 8 7 )

In the presence of excess substrate the flash of light produced is proportional to the quantity of luciferase in the reaction mixture. The light is recorded as candela (cd) the SI unit of luminous intensity. The Promega luciferase assay system is optimised to emit stable luminescence with a half-life of 5 h. To confirm that the relationship between luciferase enzyme concentration and light emitted was linear under the assay conditions used, a range of serial dilutions of commercially obtained luciferase was made, ranging from 10 ng mf^ to 0.15 fg m l'\ and assayed. Figure 3.8 shows that within the range tested, a linear relationship exists between luciferase

Chapter 3

3.3.2 The CAT reporter gene and assay

CAT is a product of an E. coli gene that confers resistance to the antibiotic chloramphenicol. CAT inactivates chloramphenicol by catalysing the formation of the mono- and diacetylated derivatives 1-acetate chloramphenicol, 3-acetate chloramphenicol and 1,3-acetate chloramphenicol. When subjected to silica thin layer chromatography (TLC) under optimal conditions (see section 2.2.4), the mono- acetylated isoforms and the diacetylated form of chloramphenicol can be distinguished by their mobility from each other and from unmodified chloramphenicol (Figure 3.9).

To define the relationship between enzyme activity and the digitised data (see section 2.2.4), a range of two-fold serial dilutions of purified commercially obtained CAT in 0.25 M Tris-HCl was prepared and assayed. Following the TLC step, the plates were dried and scanned in a phosphorimager to obtain digitised data, and the data analysed using ImageQuant™ software. The digitised data are represented as 5 peaks (Figure 3.9). Peaks 1 and 2 represent residual non-acetylated chloramphenicol, whereas peaks 3-5 represent 1-acetate chloramphenicol, 3-acetate chloramphenicol and 1,3-diacetate chloramphenicol products respectively (Gorman et a l, 1982). Levels of the mono-acetylated form of chloramphenicol (peaks 3 and 4) correlate directly with enzyme concentration (Figure 3.10a). The diacetylated form of chloramphenicol (peak 5) is only present at high enzyme concentrations. In this and subsequent assays, this form was only observed in control experiments using commercially obtained CAT; it was never observed using extracts of transfected parasites. These observations were interpreted as suggesting that that conversion of

total available substrate has been converted into the mono-acetylated form, in other words, under conditions of enzyme excess. Thus, when the CAT activity, measured as the sum of the [^"^C] activity associated with peaks 3, 4 and 5, is plotted against CAT enzyme concentration, the relationship is seen to plateau as the diacetylated form becomes detectable (Figure 3.10b). However, when chloramphenicol is presumably still in excess at the end of the 1 h reaction, as shown by high values for peak 2 (lanes 6-10; Figure 3.9), there is a linear relationship between enzyme concentration and total CAT activity. The CAT activity detected in transfected parasite cultures does not include the generation of diacetylated chloramphenicol. Thus, CAT enzyme activity detected in transfected parasite culture material directly reflects the enzyme concentration. If the effect of transcript stability is considered to be minimal, then CAT enzyme activity can be considered to represent the activity of the promoter region driving expression.

3.3.3 Validation of a dual expression system

To validate the use of two reporter gene cassettes on a single vector to control for variations in transfection efficiency, it was important to establish whether there exists a constant relationship between the activity of the two reporter genes over a range of input DNA. A P. falciparum culture at 10% parasitaemia was transfected with 100 |Lig, 75 p g or 50 p g of pCaRp33L. Luciferase activity was assayed 24 h post transfection, and CAT activity 46 h post transfection, each assay being performed in duplicate. Construct pHCl-CAT was used as a positive control for CAT activity and a negative control for luciferase activity, whilst pHEP.luc was used as a positive

Chapter 3 The activity of each reporter gene dropped as the amount of DNA used in the transfection was reduced (Figure 3.11). To calculate the ratio of luciferase to CAT activity at each DNA concentration, the CAT activity value was divided by that of

luciferase and the standard deviation calculated (Figure 3.12). The average ratio of reporter gene activity (CAT/luciferase) obtained for each value of input DNA (50, 75 and 100 p-g) was 0.76 with a standard deviation of 0.11. The CAT data showed the largest degree of variation (figure 3.11). This is perhaps not surprising as the CAT assay is a multi-step process rendering it less direct than the luciferase assay and more susceptible to inconsistencies induced during manipulation of each sample.

In conclusion, the ratio of reporter gene activity was shown to be substantially independent of the amount of input DNA used in transfection. The use of different amounts of DNA means that there is not the same amount of plasmid DNA present and transcriptionally active in the target nuclei of each sample assayed. Therefore, the ratio of reporter gene activity detected can be considered to be independent of DNA input and transfection efficiency. The ratio of reporter gene activity can also be used to normalise data for quantitative comparison between constructs in parallel experiments and from transfection to transfection.

3.4 GENE EXPRESSION IN A VARYING EPISOMAL CONTEXT