Passage
Trypanosoma brucei is a eukaryotic parasite that causes sleeping sickness in Sub-Saharan Africa. The cell cycle of T. brucei differs from that of higher eukaryotes. For example, the chromatin does not condense during mitosis, and the nuclear envelope remains intact. In addition, the nucleus divides before cell division, and the cell divides along the longitudinal axis (Figure 1) .
Figure 1 Mitosis in T. bruceiA culture of T. brucei was arrested in S phase of the cell cycle by overnight incubation with the reversible inhibitor hydroxyurea. The cells were then washed, and their progression through the cell cycle was monitored by flow cytometry and microscopy. A subset of the culture was first exposed to the drug VX-680, which arrests cells upon completion of metaphase. The results are shown in Figure 2.
Figure 2 Percentages of a population of T. brucei cells in each phase of the cell cycle, measured at different time points in the (A) absence and (B) presence of VX-680
Adapted from Li Z, Umeyama T, Wang CC. The Aurora Kinase in Trypanosoma brucei plays distinctive roles in metaphase-anaphase transition and cytokinetic initiation. PLoS Pathog. 2009;5(9) :e1000575.
-Why were T. brucei cells incubated with hydroxyurea overnight and washed afterward?

Question

Passage
Trypanosoma brucei is a eukaryotic parasite that causes sleeping sickness in Sub-Saharan Africa. The cell cycle of T. brucei differs from that of higher eukaryotes. For example, the chromatin does not condense during mitosis, and the nuclear envelope remains intact. In addition, the nucleus divides before cell division, and the cell divides along the longitudinal axis (Figure 1) .

Figure 1 Mitosis in T. bruceiA culture of T. brucei was arrested in S phase of the cell cycle by overnight incubation with the reversible inhibitor hydroxyurea. The cells were then washed, and their progression through the cell cycle was monitored by flow cytometry and microscopy. A subset of the culture was first exposed to the drug VX-680, which arrests cells upon completion of metaphase. The results are shown in Figure 2.

Figure 2 Percentages of a population of T. brucei cells in each phase of the cell cycle, measured at different time points in the (A) absence and (B) presence of VX-680
Adapted from Li Z, Umeyama T, Wang CC. The Aurora Kinase in Trypanosoma brucei plays distinctive roles in metaphase-anaphase transition and cytokinetic initiation. PLoS Pathog. 2009;5(9) :e1000575.
-Why were T. brucei cells incubated with hydroxyurea overnight and washed afterward?

Quizplus · Accepted Answer

11ecba2d_101a_d024_a3bf_29fae4b037d1_MD0008_00 The cell cycle consists of several distinct phases, and a randomly selected population of cells will most likely contain a subset of cells in each phase at any given time. As one subset of cells exits a phase, another subset progresses into that phase. The overall effect is that the total number of cells in each phase is approximately constant over time, making it difficult to monitor progress through the cycle. For this reason, cell cycle studies are often carried out in cell populations that have been synchronized (ie, experimentally brought to the same stage of the cell cycle). A population of cells may be synchronized by incubation with a chemical that reversibly blocks a cell cycle process, arresting cells in the corresponding phase. After all cells have been arrested, the chemical is washed away and the cells reenter the cycle together. The passage states that a DNA replication inhibitor (ie, hydroxyurea) was used to arrest T. brucei cells in S phase, after which these cells were washed to remove the inhibitor and cell cycle progression after release was monitored. The purpose of this protocol was to ensure that most cells were in the same phase at each time point measured. (Choice B) Although the DNA replication inhibitor arrests cells in S phase by inhibiting DNA synthesis, the purpose of using this inhibitor was to synchronize T. brucei cells, not to observe the effect of inhibiting DNA synthesis on cell cycle progression. (Choice C) Because cell growth occurs during G₁ and G₂ (but not S) phase, the DNA replication inhibitor is unlikely to alter the size of cells because it arrests cells only in S phase. (Choice D) Promoting synthesis of the proteins needed for entry into mitosis (eg, cyclins) would most likely increase, not arrest, the rate of progression through the cell cycle. The DNA replication inhibitor arrests the cells in S phase. Educational objective: A random population of cells will likely contain some cells in each phase of the cell cycle. Study of their progress through the cell cycle requires that these cells first be brought to the same phase of the cell cycle (ie, synchronized). These synchronized cells can then be monitored when they are released and progress through the cycle together.

Passage Trypanosoma Brucei Is a Eukaryotic Parasite That Causes Sleeping Sickness