Question

Plant Physiology

suppose you suspect your gene of interest is expressed during M -phase, what type of experiment(s) might you design to prove your hypothesis?

Answer 1

First we can synchronise and sort cells in G1, S, G2 and M phase by using Flow cytometer. Here first we stain our cell with propidium iodide (PI) which binds to DNA of cell. (More DNA = intense peak because of more attachment of PI). We can sort cells at small peak G1( no DNA replication) S phase (a progression of peak because start of DNA replication) Doubly intense peak at the boundary of G2-M phase because here intensity of PI will become double which signifies end of S phase i.e. DNA doubling or replication is complete.

Now we have a cell population which is mixture of cells either in G2 or M phase (because we have sorted on the basis of doubling of DNA hence double intensity of propidium iodide colour intensity and it will be found in cells of both the phases). Now We can choose a gene which is very specific for M phase like cdc 25 ( role in Kinetochore disassembly or anaphase promoting complex).

We can check co expression of cdc 25 and our hypothetical protein and infer that our protein is being expressed at least in late metaphase (a part of M phase).

Now we have to reject or confirm that whether expression of the hypothetical protein is happening in other phases of cell cycle or not. This we can check by checking expression of our hypothetical protein again by flourescently labelling it in cells by flow cytometer in G1, and S phase cells.

Another strategy will be by treating cells with colchicine and arresting cells in metaphase ( inhibits microtubule polymerization and thus assembly of the mitotic spindle). When colchicine is added to cultured cells, the cells enter mitosis and arrest with condensed chromosomes.

Now we can take such cell cycle arrested cells of Metaphase and look for expression of our hypothetical protein either by observing coexpression pattern with phosphorylated H3 (it has a role in chromatin condensation apart from other epigenetic roles).

A third strategy will be of Global transcriptome analysis at each time point of cell cycle (G1, S,G2 and M). We can optimise timing of a phase of cell cycle by harvesting cells at different time points. Ex if we know that cell completes its's cycle in 24 hour then based on our knowledge of cell cycle we can isolate mRNA at 3H, 6H 9 h and so on. and see the expression pattern of our hypothetical protein.