In the body system ofÂ Drosophila melanogaster,Â different enzymes perform different functions. For instance, enzyme DNase II is involved in the degradation of ingested DNA in phagolysosomes. Therefore, mutation or absence of this enzyme would result into increased ingested DNA in phagocytic cells. The researchers in this paper were dealing with the effects of reduced or mutated DNase II activity on the immune system ofÂ Drosophila melanogaster.Â They wanted to determine what happens when the activity of DNase II inÂ Drosophila melanogaster isÂ reduced or eliminated.
The hypothesis in this study was, â€œsevere reduction of DNase II activity would result in diminished immune function and viabilityâ€. In this study, the researchers used RNAi to knock down enzyme DNase II fromÂ Drosophila melanogaster.Â Additionally, after knocking down this enzyme, they infected the DNase II-deficient flies with gram-negative bacteria to test the level of infection.
The techniques used in this study varied even though they worked in harmony to achieve the desired results. The activity of the DNase II enzyme derived from the flies was determined by use of radical diffusion assay. Other techniques used included construction of RNAi vector by cloning its fragments into pGEM â€“T vector. Inverse Polymerase Chain Reaction (PCR) was used to verify the site of integration and to sequence the region adjacent to the RNAi transposon. Quantitative real-time PCR was then performed to synthesis cDNA. The other test involved was testing survival of the flies after knocking out the DNase II enzyme and the subsequent infection of gram-negative bacteria. After this, the researchers generated polyclonal antibodies against the carboxyl terminal of the enzyme and carried out a western blot to determine the amount of DNase II enzyme and mRNA in the DNase II-deficient flies. The final test was to determine the effects of DNase II-RNAi expression on dDNase II levels and hemocyte numbers by use of immunoflourescence microscopy.
The researchers found interesting results. Contrary to the common belief that mutation or degradation of DNase II inÂ Drosophila melanogasterÂ leads to increase in un-degraded DNA in phagocytic cells, they established this statement is null. The study revealed that this is not the case. Despite the fact that these researchers knocked out enzyme DNase II from the flies, the amount of un-degraded DNA in phagocytic cells remained low. One of the explanations put forward here is that, residue effect of nuclease activity in DNase II-deficient flies is sufficient to degrade DNA in these phagocytic cells.