When normal human fibroblasts are cultured in medium containing fetal bovine ser

Question

When normal human fibroblasts are cultured in medium containing fetal bovine serum that contains growth factors, they divide with an average generation time of approximately 22 hours (M 1 hr, G1 100 hr. S 6 hr, G2 5 hr). To determine the effects of serum deprivation on cell cycle distribution, cells were incubated for 48 hours in medium with or without serum. At the end of this incubation, cells were harvested and stained with propidium iodide, which binds to DNA and fluorescences when exposed to ultraviolet light. The stained cells were analyzed for DNA content (fluorescence) in a flow cytometer. The results with serum are shown in the Figure la. If deprived of serum, the cells stop proliferating and Go, a quiescent state (Figure lb. In the second experiment, cells were deprived of serum for 48 hours and then treated either serum alone or serum plus cycloheximide (CHX), an inhibitor ofprotein synthesis. At various time after treatment, RNA was isolated from the cells. Equal amount of total cellular RNA from each sample were analyzed by gel electrophoresis and Northern blotting to detect c fos mRNA and quantify the level of cofos mRNA. The c-Fos protein is involved in regulating cell proliferation. The results of this secondexperiment are shown in Figure 2. indicates serum alone and indicates serum plus CHX). 10 15 20 10 15 20 Fluorescence (relative units) Fluorescence (relative units Figure la. With scrum Figure lb. Without serum Time after serum addition (hours) Figure 2. cfos mRNA.

Explanation / Answer

B. The thymidine labelled cells on incorporation during the replication, will show the radioactivity only during its S- phase (synthesis phase, wherein DNA is being replicated).

The histogram labelled as X,Y and Z show the phases of G1, S and G2/M respectively.

Thus, radioactivity will be seen during Y phase only, therefore the answer will be option b.

C. By comparing Lane 3 and Lane 5, there is decrease in the length. There could be two conclusions from this, either there is shortening of Poly(A) tails or there must be some process in the inital steps that induces the elimination of the c-fos mRNA from cell. Thus, from this inference, option d proves to be suitable. The splicing of c-fos pre-mRNA requires an unstable splicing factor.

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