This Data Analysis Problem does not appear in the textbook.

Sources: Warner J. R., P. M. Knopf, A. Rich. 1963. A multiple ribosomal structure in protein synthesis. Proc. Natl. Acad Sci. USA 49(1): 122–129.

Penman S., K. Scherrer, Y. Becker, J. E. Darnell. 1963. Polyribosomes in normal and poliovirus-infected HeLa cells and their relationship to messenger-RNA. Proc Natl Acad Sci USA 49(5): 654–662.

Corresponding chapter(s) in the textbook: Chapter 10

Review the following terms before working on the problem: [3H]leucine labeling, translation, sucrose density gradient centrifugation, UV absorption, ribosomes, polysomes

Experiment

Rabbit reticulocytes (immature red blood cells, graph A), HeLa cells (human cancer cells, graph B), and poliovirus-infected HeLa cells (C) were pulse-labeled for 1 minute with [3H]leucine. The cell membranes were then dissolved with a detergent, and the cell extracts were sedimented through a sucrose gradient. Identical volumes of solutions were collected from the bottom of the gradients, transferred into test tubes, and the radioactivity and ultraviolet (UV) absorption (A260) were measured for each fraction.

Figure

Sucrose density gradient centrifugation of cell extracts. (Arrow indicates direction of centrifugation.)

Questions

1. What was the goal of [3H]leucine labeling in this experiment?

2. What does the UV absorption peak in fractions 20–25 correspond to?

3. What does the radioactivity at the top of the gradient (fractions 27–28) correspond to?

4. What do the other radioactivity peaks, closer to the bottom of centrifuge tubes, correspond to?

5. Interpret the differences between the radioactivity curves in the three graphs.

6. The radioactivity and UV absorption peaks between fractions 10 and 20 completely disappear in virus-infected cell extracts (compare Graphs B and C). Suggest an explanation for this result.