1 MCB 502A-2015. Lecture #11. The Cell Cycle. The prokaryotic cell cycle Helmstetter's “baby machine” Is there a way to synchronize a culture without, actually, synchronizing it? This sounds like another paradox, but technically it is in fact not a problem with E. coli or any other cells that can be fractionated in gradients or can permanently stick to surfaces. Instead of synchronizing bacteria, Helmstetter fractionated them according to their age at the time of pulse-labeling with a radioactive precursor. Helmstetter added the label to an asynchronous growing culture and then terminated the labeling by filtering bacterial suspension and flushing the filter with an unlabeled growth medium to remove the label. E. coli strain that he has been using was known to attach permanently to this type of filters. Helmstetter then inverted the filter and dripped the same growth medium, but now without the label, onto the filter, collecting the eluate with whatever cells were able to elute from the filter. The original bacteria are attached too tightly to the membrane and do not elute under these conditions, but the newly-born bacteria do, if they do not touch the membrane. By collecting 4-minute fractions, Helmstetter in effect fractionated bacteria by their age at the time of pulse-labeling. His reasoning was as follows (Figure 1 of Helmstetter-67): if a bacterium was at the end of its cycle at the time of labeling, then its daughter cell would be eluted right away; if bacterium was in the middle of its cell cycle, then its daughter would be eluted after half the cell cycle time; whereas if the cell was just born at the time of labeling and attachment, its daughter cell would be eluted after a period of time equal to the division time under these growth conditions. Does the expected number of cells eluted with time follow a straight line? Counterintuitively, the theoretical elution curve for an exponentially-growing culture is a saw-tooth pattern: (Figure 2B of Helmstetter-67). Indeed, at the very beginning, one is eluting daughters of cells that were ready to divide before filtration, while at the end of a cycle, one is eluting daughters of cells that were just born when the culture was attached to the membrane. The ratio of a cell that is about to divide to its two daughters right after division is 1:2. In other words, in any growing culture of cells dividing by fission, newborn cells are almost twice as frequent as cells that are about to divide. Therefore, the plot of the relative frequency of cells as a function of their age along the division cycle is a line decreasing from 2 to 1 (Figure 2A of Helmstetter-67). Helmstetter in fact observed the derivatives of the expected cell number curve when he grew his E. coli in four different media with various carbon sources, supporting division times of 45, 60, 80 and 120 minutes: (Figures 4 Helmstetter-67). His curves were not going down to abscissa because (he suspected) some of the newborn cells after the first generation were stuck on the membrane, and so, later on, there was an overflow of these cells. It was also possible that cells were just detaching with time from the membrane (due to cell wall renovation, for one). Concept-in-the-box. Synchronization without interference with growth of the culture is achieved by fractionation. In general, the newly-born cells are two times smaller than the cells that are about to divide, and therefore can be separated from the latter by gradient centrifugation as, correspondingly, the slowest- and the fastest-sedimenting fractions. This approach works best for bacteria and yeast, and in general for organisms in which the hard cell envelope keeps the cell shape constant. Cultured animal cells can be synchronized due to the fact that they become loosely attached to their substrate during mitosis. Helmstetter used yet another fractionation trick — fractionation by time, eluting only the newly-born cells. However, the real power of his technique was in the way he combined this fractionation with the preceding pulse labeling. Instead of experimenting with the fractionated cells, he just measured their radioactivity that reflected incorporation in their parents at a specific part of the parental cell cycle. By the way, he later patented the same synchronization approach for human lymphocytes, so it is not only applicable to bacteria…2 Now, let us plot the rate of synthesis of a hypothetical macromolecule as a function of the cell age. If the macromolecule is synthesized throughout the cell cycle (like a ribosome or a protein or a cell wall), its rate of synthesis will increase continuously and exponentially. Therefore, if we pulse-label the macromolecule and then follow the radioactivity in the eluted cells for several generations, we will observe a straight declining line in a semilog plot. The plot is semi-log because of the two-fold dilution every generation of the total radioactivity in the attached parental cells: (Figure 3AB Helmstetter-67). If the macromolecule in question is again synthesized continuously, but at a constant rate, and this rate of synthesis doubles halfway through the cell cycle, we will plot the step in relative synthesis rate as a function of cell age, and a descending step function for the relative radioactivity as a function of the elution time. At last, if the macromolecule in question is synthesized during the central 50% of the cell cycle, its plotted rate of synthesis will resemble a haystack (starting and returning to zero), and the elution profile will be a collection of two-fold-diminishing haystacks. This is what Helmstetter observed when he pulsed-labeled DNA in his cells, growing in four different media, and counted specific radioactivity of the eluted cells: (Figures 5 Helmstetter-67). “Haystacks” in the acetate medium (division time 120 minutes) indicate that most DNA synthesis happens during the central 60 minutes of the cell cycle (defined here as the period between two consecutive cell divisions). “Steps” in the glucose medium indicate that DNA synthesis rate doubles halfway through the cell cycle, suggesting the new initiation coinciding with termination of the current chromosome replication round. As you see, these elution patterns allow one to accurately measure in diverse growth conditions: 1) the generation time; 2) the length of the DNA synthesis period; 3) the relationship between the two periods. These measurements can be
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