In our laboratory we investigate the regulation of intestina

In our laboratory, we investigate the regulation of intestinal stem cells in fruit flies by a transcriptional repress or called Escargot (Esg). We identified a gene called Amun as a potential target of Esg, and hypothesized that if we inhibited Esg function within cells, the transcription of Amun would be de-repressed, and the amount of Amun mRNA within cells would increase. How can we be sure that this accumulation in Amun mRNA is due to increased transcription and what might be another alternative hypothesis that, if true, would lead to the same observation?

Additional tools that we can use for this experiment are 1) antibodies for immunoprecipitation of RNA pol II, 2) \"RNA sequencing\", and 3) transcriptional inhibitor.

Solution

Answer) As we know that any form of biological reaction is controlled by two things; activity and quantity. to be sure for the desired outcome, we need to standardize the quantity and then regulate the activity.

To be sure that this accumulation in Amun mRNA is due to increased transcription, you will need to assess the level of Amun mRNA in absence of Esg, in presence of Esg and in presence of non-functional Esg. because a Non-functional Esg might not be regulating the transcriptional repression via Amun gene only but it might have multiple targets. if you see the similar level of transcript in absence of Esg and in presence of non-functional Esg, then it is due to increased transcription which was repressed by Esg repressor targeting only Amun gene.

and please keep this point in your mind that more Amun mRNA will be synthesized if there is increased transcription, but at the same time Amun mRNA may be accumulated due to inhibited/slow/poor mRNA degradation.

Another hypothesis is that \"increased transcription do not lead to accumulation of Amun mRNA, but it is due to non-translation and slow degradation of Amun mRNA which was earlier mediated by functional Esg\" and to test this hypothesis, you will need to perform standardization of transcriptional repressor in terms of amount of repressor and the time duration. only then you can be sure that this is not happening the other way.