The life cycle of eukaryotic cells can generally be divided into four stages and a typical cell cycle is shown in Figure \(\PageIndex{13}\). When a cell is produced through fertilization or cell division, there is usually a lag before it
undergoes DNA synthesis (replication). This lag period is called Gap 1 (G1), and ends with the onset of the DNA synthesis (S) phase, during which each chromosome is replicated. Following replication, there may be another lag, called Gap 2 (G2), before mitosis (M). Cells undergoing meiosis do not usually have a G2 phase. Interphase is as term used to include those phases of the
cell cycle excluding mitosis and meiosis. Many variants of this generalized cell cycle also exist. Some cells never leave G1 phase, and are said to enter a permanent, non-dividing stage called G0. On the other hand, some cells undergo many rounds of DNA synthesis (S) without any mitosis or cell division, leading to endoreduplication. Understanding the control of the cell cycle is an active area of research, particularly because of the relationship between
cell division and cancer. Measures of DNA content and chromosome contentThe amount of DNA within a cell changes following each of the following events: fertilization, DNA synthesis, mitosis, and meiosis (Fig 2.14). We use “c” to represent the DNA content in a cell, and “n” to represent the number of complete sets of chromosomes. In a gamete (i.e. sperm or egg), the amount of DNA is 1c, and the number of chromosomes is 1n. Upon fertilization, both the DNA content and the number of chromosomes doubles to 2c and 2n, respectively. Following DNA replication, the DNA content doubles again to 4c, but each pair of sister chromatids is still counted as a single chromosome (a replicated chromosome), so the number of chromosomes remains unchanged at 2n. If the cell undergoes mitosis, each daughter cell will return to 2c and 2n, because it will receive half of the DNA, and one of each pair of sister chromatids. In contrast, the 4 cells that come from meiosis of a 2n, 4c cell are each 1c and 1n, since each pair of sister chromatids, and each pair of homologous chromosomes, divides during meiosis.  Beyond immunofluorescence, cell cycle studies are another popular flow cytometetric technique. The Circle of Life Cellular division, or cell cycling, occurs in two distinct phases, Interphase and Mitosis. Interphase consists of the G1, S and G2 phases:
Mitosis occurs in the M phase in a four-step process (prophase, metaphase, anaphase, telophase) which results in cell division and normal dna content. G0 is a somewhat controversial stage as some investigators do not believe that it truly exists. Cells in G0 have exited mitosis and are quiescent. These resting cells may be reactivated and enter the G1 phase upon receipt of the appropriate stimuli. Propidium Iodide Staining Propidium Iodide is a typical cell cycle stain. The dye passes through a permeabilized membrane and intercalates into cellular DNA.The intensity of the PI signal, then, is directly proportional to DNA content. In the above image, we see three different examples of cell cycle analysis. In the first image (upper left), we see a typical cellular state with the majority of cells in the G0/G1 phase (tallest peak). The rightmost peak on the histogram shows the cells in the G2/M state. The area -between these peaks indicates cells within S-phase. The following two histograms show the cells after various drug treatments. The upper-right image shows the cells being arrested in G2. The lower-left histogram shows a marked increase in apoptosis as indicated by the substantial decrease in DNA content (those events showing sub-G0 DNA content). The lower-right histogram shows an overlay of the three histrograms in these states. As you can probably imagine, it can be very difficult to determine quantitative measurements of each of the cell cycle phases by simple gating-analysis of the cytometric data (Here are some data files if you care to give it a try). A better approach is to use a modelling program. The Flow Core’s FACSCalibur is equipped with Verity Software House’s Modfit LT. This program, and others like it, utilizes peak fitting techniques to automatically model the pi data and provides the desired quantitative data. Each iteration also provides a statistical analysis of the modelling effort to provide an indication of accuracy. A typical such modelling can be seen below. Staining Techniques For your convenience, we have placed two common Cell Cycle staining protocols on the core web site. The first is a simple ethanol-fixation technique. This protocol produces excellent results and is the most often used amongst flow core clientele. The second, involves a hypotonic lysis of the cell so that free nuclei are analyzed. This procedure can also produce good results and should be considered if you experience problems with the fixation technique. BRDU While PI staining provides a very precise image of the cellular state with regards to cell cycle, it really provides no information regarding the kinetics of cell cycle. BRDU staining is a popular method for beginning to look at the kinetics of the situation. Bromodeoxy-uridine (BRDU) is similar to the DNA precusor, thymidine. During S-phase, in the presence of BRDU, the cells with incorporate BRDU during the S-phase. After a targetted incubation period, fluorescently-labelled antibodies specific to BRDU can be added to provide an indication of which cells have entered the cell cycle during the incubation period. This technique is often used with PI staining in order to provide a clearer image of cell cycle. The image above shows cells labelled with PI and BRDU-Fitc. Staining Techniques Wrapping Up In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below. After receiving this I will provide you with access to the next module.
What is the S phase of cell cycle?S phase is the period of wholesale DNA synthesis during which the cell replicates its genetic content; a normal diploid somatic cell with a 2N complement of DNA at the beginning of S phase acquires a 4N complement of DNA at its end.
Which of the following occurs during S phase?In the S phase (synthesis phase), DNA replication results in the formation of two identical copies of each chromosome—sister chromatids—that are firmly attached at the centromere region. At this stage, each chromosome is made of two sister chromatids and is a duplicated chromosome.
What is the S phase in interphase called?Interphase is defined by three stages: the first gap phase (G1), the synthesis (S) phase, and the second gap (G2) phase.
What happens in S phase?The S phase of a cell cycle occurs during interphase, before mitosis or meiosis, and is responsible for the synthesis or replication of DNA. In this way, the genetic material of a cell is doubled before it enters mitosis or meiosis, allowing there to be enough DNA to be split into daughter cells.
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Based on the graph, which of the following labeled phases corresponds to s phase of the cell cycle?
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