Question

You are a graduate student studying an inducible transcription factor called X. You make a cellular lysate and carry out a series of centrifugation steps at increasing speeds to localize X. You find that prior to induction, X is the supernatant following a fourth, very high speed ultracentrifugation step; however, after induction it is located in the pellet after only one relatively low speed ultracentrifugation step. What might this tell you about how X is regulated? What other techniques might you carry out to confirm your results?

Answer 1

This indicates that X is present in the cytoplasm before induction and localises to the nucleus upon induction to carry out the transcription of its target genes.

Reason: ultracentrifugation is a process of applying various able magnitudes of centrifugal force (here, to the lysed cells) - the centrifugation force increases each time the lysate undergoes centrifugation. The heavier components of the cells will pellet down at lower speeds owing to their higher molecular weight while the light one will require comparatively higher speeds to sediment/ pellet out. Following is the general order in which the cell component pellet out:-

> Nucleus

> Most other organelles like mitochondria, peroxisomes, chloroplasts, etc

> Microsomes ( disrupted vesicles of the endoplasmic reticulum)

> Cytoplasmic components

An immunostaining of the cells using antibody specific for factor X, before and after induction, and help you confirm its localisation in the 2 cases.

Also an EMSA, if the target genes for X are known would help. Binding to DNA would be observed in case of induction of while the no binding would be observed when not induced. But this is not confirmation of its localises in the cytoplasm.