Information

DNA content doubling in interphase


Why does the DNA content of a cell get doubled in interphase? Why doesn't it become tripled or quadrupled? What's stopping it from doing so?


So in mitosis, the cell has to split itself into two cells; each daughter cell has a functional genome that may again split into more daughter cells. The cell replicates the DNA before dividing, so the error in replicating 3x or 4x is that upon division, the daughter cells will have more DNA than the initial cell, and every generation will have more DNA than the last. One replication ensures both daughter cells receive one functional genome. We can see the result of extra DNA in cases like Down Syndrome (trisomy of chromosome 21).

The cell controls this process by controlling the cyclins present in the cell temporally, namely cyclin-A and cyclin-E when we talk about DNA replication in S-phase,

From wikipedia,

Cyclin A resides in the nucleus during S phase where it is involved in the initiation and completion of DNA replication. As the cell passes from G1 into S phase, cyclin A associates with CDK2, replacing cyclin E. Cyclin E is responsible for initiating the assembly of the pre-replication complex. This complex makes chromatin capable of replication. When the amount of cyclin A/CDK2 complex reaches a threshold level, it terminates the assembly of the pre-replication complex made by cyclin E/CDK2. As the amount of Cyclin A/CDK2 complex increases, the complex initiates DNA replication.

Cyclin A has a second function in S phase, in addition to initiating DNA synthesis, Cyclin A ensures that DNA is replicated once per cell cycle by preventing the assembly of additional replication complexes. This is thought to occur through the phosphorylation of particular DNA replication machinery components, such as CDC6, by the cyclin A/CDK2 complex. Since the action of cyclin A/CDK2 inhibits that of cyclin E/CDK2, the sequential activation of cyclin E followed by the activation of cyclin A is important and tightly regulated in S phase.

Source: Cyclin A


What is G1 Phase?

Gap 1 or G1 phase is the first cell growth phase of the interphase of the cell cycle. Significant development processes take place within the cell during the G1 phase. The cell size will increase due to the extensive synthesis of proteins and RNA. Synthesis of proteins and RNA is required prior to S phase where the replication of DNA takes place. Proteins synthesized during the G1 phase mainly include histone proteins, and the majority of RNA synthesized is mRNA. Histone proteins and mRNA participate in the S phase for DNA replication.

The duration of the cell cycle varies according to the type of organism. Some organisms have a longer G1 phase before entering the S phase while other organisms may have a shorter G1 phase. In humans, a typical cell cycle runs for 18 hours. Out of the total time of the cell cycle, G1 phase normally takes 1/3 of the time. However, this time can change due to certain factors. These factors are referred to as growth factors, and some of them are cellular environment, availability of nutrients such as proteins and specific amino acids and cellular temperature. The temperature mainly affects the proper growth of the organism, and this value varies from organism to organism. In humans, the optimum temperature for cellular growth is roughly 37 0 C.

Figure 01: Cell Cycle

The cell cycle regulatory mechanism controls the G1 phase. During the regulation, the control of duration and coordination between other phases take place. G1 phase is considered to be an important phase because it is the point which determines the fate of the cell. In this phase, the cell decides whether it proceeds with the rest of the phases of the cell cycle or leave the cell cycle. If the cell receives a signal to keep it at an un-dividing stage, the cell will not enter into the S phase. It will move into the dormant phase called G0 phase. G0 phase is a state of cell cycle arrest.


Materials and Methods

Plant material

The following wheat (Triticum aestivum) genotypes were used:AABBDD, 2n=6×=42, cv. Chinese spring with the addition of rye(Secale cereale L. cv. Imperial) chromosome pairs, 5R or 1R, and translocation lines where the long arm of wheat chromosome 1A or 1D is replaced by the short arm of rye chromosome 1R (1A 1 /1R S )and (1D 1 /1R S ), respectively. Wheat lines (cv. Bobwhite)were transformed with the plasmid pAHC25, containing the Gus reporter gene, by particle bombardment using the Biolistics PDS 1000/He device(Abranches et al., 2000). Seeds were germinated for 4 days at 24°C on filter paper soaked in water alone or water containing 80 μM 5-azacytidine (5-AC, Sigma) or water containing 15 μM Trichostatin A (TSA — Sigma) (diluted just prior to use from a 10 mM stock solution in dimethyl sulfoxide). The 5-AC was freshly dissolved in water and changed daily. The root-tips were excised and fixed in 4% (w/v)formaldehyde freshly prepared from paraformaldehyde in PEM buffer (50 mM PIPES/KOH pH 6.9 5 mM EGTA 5 mM MgSO4) for 1 hour at room temperature, then washed in TBS (10 mM Tris-HCl, pH 7.4 140 mM NaCl) for 10 minutes.

Protein extraction and immunoblotting analysis

Total root proteins were extracted by homogenising roots in SDS sample buffer (Laemmli, 1970) [Sample buffer: 0.125 M TRIS/HCl pH 6.8, 4% (w/v) SDS, 20% glycerol, 10% (v/v)2-mercaptoethanol, 0.002% (w/v) bromophenol blue]. Protein samples were resolved by SDS gel electrophoresis on 15% gels and transferred to nitrocellulose by western blotting (Towbin et al, 1979). The blots were probed with antibody AHP418(Serotec), which is specific for acetylated histone H4, or antibody AHP416(Serotec), which is specific for Histone H4 acetylated at lysine 12, diluted in TBS according to the manufacturer's instructions. Proteins were visualised using a secondary antibody goat anti-rabbit alkaline phosphatase, diluted 1 in 1000 in TBS.

Root sections

30 μm thick sections from root tips were sectioned using a Vibratome Series 1000 (TAAB Laboratories Equipment Ltd., Aldermarston, UK) and allowed to dry on multi-well slides (ICN Biomedicals Inc.). The slides were pre-treated by washing in 3% (v/v) Decon for 1 hour, rinsing thoroughly with distilled water. They were then coated with a freshly prepared solution of 2%(v/v) 3-aminopropyl triethoxy silane (APTES, Sigma) in acetone for 10 seconds and activated with 2.5% (v/v) glutaraldehyde in phosphate buffer for 30 minutes, rinsed in distilled water and air dried.

In situ hybridisation on wheat root sections

The tissue sections were permeabilised by incubation with 2% (w/v)cellulase (Onozuka R-10) in TBS for 1 hour at room temperature, washed in TBS for 10 minutes, dehydrated in an ethanol series of 70% and 100% and air dried. Root sections from wheat transgenic lines were additionally treated with RNAse(100 μg/ml) for 1 hour at 37°C, washed in 2×SSC (20×SSC: 3 M sodium chloride, 300 mM trisodium citrate, pH 7.0) and dehydrated as described above. Genomic in situ hybridisation and generation of total genomic probe was performed according to Schwarzacher et al.(Schwarzacher et al., 1992)and Abranches et al. (Abranches et al.,1998). The hybridisation mixture contained 50% deionised formamide, 20% dextran sulphate, 0.1% sodium dodecyl sulphate, 10%20×SSC, 200 ng of rye genomic DNA sonicated to 10-12 kb fragments as a probe and 1 μg of sonicated salmon sperm as blocking DNA. Fluorescence in situ hybridisation was used to visualise the transgenes on wheat root sections, using pHAC25 DNA (200 ng) as a probe. Probes were labelled with digoxigenin-11-dUTP (Boehringer Mannheim Corp. Indianapolis, IN) or biotin-16-dUTP (Boehringer Mannheim) by nick translation. Denaturation of the hybridisation mixture was carried out at 95°C for 5 minutes, cooled in ice for another 5 minutes and immediately applied to the sections. Target DNA denaturation was carried out in a modified thermocycler (Omnislide Hybaid LTD., Long Island, NY) at 78°C for subsequent hybridisation at 37°C overnight. Post-hybridisation washes were carried out using 20% formamide in 0.1SSC at 42°C.

BrUTP incorporation into tissue sections

For transcription analysis, the procedures followed are those described previously (Thompson et al.,1997 Abranches et al.,1998). Briefly, vibratome sections were cut in a Modified Physiological Buffer (MPB: 100 mM potassium acetate, 20 mM KCl, 20 mM Hepes 1 mM MgCl2 1 mM ATP (disodium salt, Sigma) in 50 mM Tris, pH 8.1%(v/v) 1% (v/v) thiodiglycol (Sigma), 2 μg/ml aprotinin (Sigma) and 0.5 mM PMSF (Sigma). To improve nuclear transcription as opposed to nucleolar transcription, 1% BSA was added to the MPB buffer. The tissue sections were transferred to a tissue-handling device(Wells, 1985) for subsequent ease of handling. The permeabilisation was done by a very brief treatment (10 seconds) with 0.05% Tween 20 in MPB. The transcription mix consisted of 50μM CTP (sodium salt, Pharmacia), 50 μM GTP (sodium salt, Pharmacia), 25μM BrUTP (sodium salt, Sigma), 125 μM MgCl2, pH 7.4 with KOH) 100 U/ml RNA guard (Pharmacia) made up in MPB. The tissue was incubated with the transcription mix for 5 minutes and then fixed in 4% formaldehyde in PEM as described above. After fixation, the sections were washed in TBS, then in water and finally removed from the tissue-handling device and placed onto activated APTES-treated slides.

Immunodetection

Probes labelled with digoxigenin were detected by an anti-digoxigenin antibody conjugated to FITC (Boehringer Mannheim Corp., Indianapolis, IN), and biotin-labelled probes were detected with extravidin-cy3 (Sigma, Chemical Co.). Both antibodies were diluted in 3% BSA in 4×SSC/ 0.2% tween-20(Sigma), and the antibody incubations were carried out in a humid chamber for 1 hour at 37°C followed by 3×5 minutes washes in 4×SSC/0.2%Tween-20 at room temperature. The detection of BrUTP incorporation involved incubation for 1 hour at room temperature with mouse anti-BrdU (Boehringer)followed by a second incubation with a secondary fluorescent anti-mouse Alexa-568 (Molecular Probes) antibody for 1 hour at room temperature. The sections were counterstained with 1 μg/ml,4′6-diamidino-2-phenylindole (DAPI — Sigma Chemical Co) for 5 minutes and mounted in Vectashield antifade solution (Vector Laboratories Inc. Burlingame, CA).

Β-Glucuronidase (Gus) assay

Gus activity was determined by testing root material by a quantitative assay as described previously (Jefferson et al., 1987), using 4-methyl umbelliferyl glucuronide (MUG) as a substrate.

Confocal fluorescence microscopy and imaging processing

Confocal optical section stacks were collected using a Leica TCS SP confocal microscope (Leica Microsystems, Heidelberg GMbH, Germany) equipped with a Krypton and an Argon laser. The microscopy data were then transferred to NIH image (a public domain program for the Macintosh by W. Rasband available via ftp from ftp://zippy.nimh.nih.gov)and composited using Adobe Photoshop 5.0 (Adobe Systems Inc., Mountain View,CA). 3D models were made from stacks of confocal sections using Object-Image[an extension to NIH image written by Vischer et al.(Vischer et al., 1994)] by drawing manually the limit of the nucleus and marking the localisation of the transgene fluorescence sites as dots. The 3D reconstruction models were visualised using Rotater (by Craig Kloeden) available from ftp://Rarn.adelaide.edu.au/rotater/rotater-3.5.cpt.hqx). Final images were printed on a Pictography P3000 printer.


Chapter 1 DNA Sequence Localization in Metaphase and Interphase Cells by Fluorescence in Situ Hybridization

This chapter describes the in situ hybridization techniques used for labeling specific sequences in chromatin fixed to slides and in suspension and discusses the procedures used to label probes with biotin, digoxigenin, and aminoacetylfluorene (AAF). The simplest and most reproducible means of labeling DNA sequence probes is by nick translation. The chapter describes the techniques for one-color fluorescent detection of these probe labels along with the techniques used for the simultaneous detection of two probes (AAF and biotin or digoxigenin, and biotin) using two different fluorochromes. DNA can be chemically modified with AAF through a chemical reaction at the C-8 carbon of guanine by the carcinogen N-acetoxy-2- aminoacetylfluorene (N-A-AAF). The AAF and biotin procedures are adapted to label nuclei in suspension for the quantitation of bound probe by flow cytometry or for analysis of nuclear organization by optical sectioning or confocal microscopy. The chapter reviews the procedure followed for suspension labeling. The procedures for DNA sequence localization in interphase and metaphase cells described in the chapter have a number of research applications.


Cell Cycle Checkpoints

The controls discussed in the previous section regulate cell cycle progression in response to cell size and extracellular signals, such as nutrients and growth factors. In addition, the events that take place during different stages of the cell cycle must be coordinated with one another so that they occur in the appropriate order. For example, it is critically important that the cell not begin mitosis until replication of the genome has been completed. The alternative would be a catastrophic cell division, in which the daughter cells failed to inherit complete copies of the genetic material. In most cells, this coordination between different phases of the cell cycle is dependent on a system of checkpoints and feedback controls that prevent entry into the next phase of the cell cycle until the events of the preceding phase have been completed.

Several cell cycle checkpoints function to ensure that incomplete or damaged chromosomes are not replicated and passed on to daughter cells (Figure 14.8). One of the most clearly defined of these checkpoints occurs in G2 and prevents the initiation of mitosis until DNA replication is completed. This G2 checkpoint senses unreplicated DNA, which generates a signal that leads to cell cycle arrest. Operation of the G2 checkpoint therefore prevents the initiation of M phase before completion of S phase, so cells remain in G2 until the genome has been completely replicated. Only then is the inhibition of G2 progression relieved, allowing the cell to initiate mitosis and distribute the completely replicated chromosomes to daughter cells.

Figure 14.8

Cell cycle checkpoints. Several checkpoints function to ensure that complete genomes are transmitted to daughter cells. One major checkpoint arrests cells in G2 in response to damaged or unreplicated DNA. The presence of damaged DNA also leads to cell (more. )

Progression through the cell cycle is also arrested at the G2 checkpoint in response to DNA damage, such as that resulting from irradiation. This arrest allows time for the damage to be repaired, rather than being passed on to daughter cells. Studies of yeast mutants have shown that the same cell cycle checkpoint is responsible for G2 arrest induced by either unreplicated or damaged DNA, both of which signal cell cycle arrest through related pathways.

DNA damage not only arrests the cell cycle in G2, but also slows the progression of cells through S phase and arrests cell cycle progression at a checkpoint in G1. This G1 arrest may allow repair of the damage to take place before the cell enters S phase, where the damaged DNA would be replicated. In mammalian cells, arrest at the G1 checkpoint is mediated by the action of a protein known as p53, which is rapidly induced in response to damaged DNA (Figure 14.9). Interestingly, the gene encoding p53 is frequently mutated in human cancers. Loss of p53 function as a result of these mutations prevents G1 arrest in response to DNA damage, so the damaged DNA is replicated and passed on to daughter cells instead of being repaired. This inheritance of damaged DNA results in an increased frequency of mutations and general instability of the cellular genome, which contributes to cancer development. Mutations in the p53 gene are the most common genetic alterations in human cancers (see Chapter 15), illustrating the critical importance of cell cycle regulation in the life of multicellular organisms.

Figure 14.9

Role of p53 in G1 arrest induced by DNA damage. DNA damage, such as that resulting from irradiation, leads to rapid increases in p53 levels. The protein p53 then signals cell cycle arrest at the G1 checkpoint.

Another important cell cycle checkpoint that maintains the integrity of the genome occurs toward the end of mitosis (see Figure 14.8). This checkpoint monitors the alignment of chromosomes on the mitotic spindle, thus ensuring that a complete set of chromosomes is distributed accurately to the daughter cells. For example, the failure of one or more chromosomes to align properly on the spindle causes mitosis to arrest at metaphase, prior to the segregation of the newly replicated chromosomes to daughter nuclei. As a result of this checkpoint, the chromosomes do not separate until a complete complement of chromosomes has been organized for distribution to each daughter cell.


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Biology 171

By the end of this section, you will be able to do the following:

  • Describe the three stages of interphase
  • Discuss the behavior of chromosomes during karyokinesis/mitosis
  • Explain how the cytoplasmic content is divided during cytokinesis
  • Define the quiescent G0 phase

The cell cycle is an ordered series of events involving cell growth and cell division that produces two new daughter cells. Cells on the path to cell division proceed through a series of precisely timed and carefully regulated stages of growth, DNA replication, and nuclear and cytoplasmic division that ultimately produces two identical (clone) cells. The cell cycle has two major phases: interphase and the mitotic phase ((Figure)). During interphase , the cell grows and DNA is replicated. During the mitotic phase , the replicated DNA and cytoplasmic contents are separated, and the cell cytoplasm is typically partitioned by a third process of the cell cycle called cytokinesis . We should note, however, that interphase and mitosis (kayrokinesis) may take place without cytokinesis, in which case cells with multiple nuclei (multinucleate cells) are produced.


Interphase

During interphase, the cell undergoes normal growth processes while also preparing for cell division. In order for a cell to move from interphase into the mitotic phase, many internal and external conditions must be met. The three stages of interphase are called G1, S, and G2.

G1 Phase (First Gap)

The first stage of interphase is called the G1 phase (first gap) because, from a microscopic point of view, little change is visible. However, during the G1 stage, the cell is quite active at the biochemical level. The cell is accumulating the building blocks of chromosomal DNA and the associated proteins as well as accumulating sufficient energy reserves to complete the task of replicating each chromosome in the nucleus.

S Phase (Synthesis of DNA)

Throughout interphase, nuclear DNA remains in a semi-condensed chromatin configuration. In the S phase , DNA replication can proceed through the mechanisms that result in the formation of identical pairs of DNA molecules—sister chromatids—that are firmly attached to the centromeric region. The centrosome is also duplicated during the S phase. The two centrosomes of homologous chromosomes will give rise to the mitotic spindle , the apparatus that orchestrates the movement of chromosomes during mitosis. For example, roughly at the center of each animal cell, the centrosomes are associated with a pair of rod-like objects, the centrioles , which are positioned at right angles to each other. Centrioles help organize cell division. We should note, however, that centrioles are not present in the centrosomes of other eukaryotic organisms, such as plants and most fungi.

G2 Phase (Second Gap)

In the G2 phase , the cell replenishes its energy stores and synthesizes proteins necessary for chromosome manipulation and movement. Some cell organelles are duplicated, and the cytoskeleton is dismantled to provide resources for the mitotic phase. There may be additional cell growth during G2. The final preparations for the mitotic phase must be completed before the cell is able to enter the first stage of mitosis.

The Mitotic Phase

The mitotic phase is a multistep process during which the duplicated chromosomes are aligned, separated, and move into two new, identical daughter cells. The first portion of the mitotic phase is called karyokinesis , or nuclear division. As we have just seen, the second portion of the mitotic phase (and often viewed as a process separate from and following mitosis) is called cytokinesis—the physical separation of the cytoplasmic components into the two daughter cells.

Revisit the stages of mitosis with The Cell Cycle & Mitosis Tutorial (webpage).

Karyokinesis (Mitosis)

Karyokinesis, also known as mitosis , is divided into a series of phases—prophase, prometaphase, metaphase, anaphase, and telophase—that result in the division of the cell nucleus ((Figure)).


Prophase (the “first phase”): the nuclear envelope starts to dissociate into small vesicles, and the membranous organelles (such as the Golgi complex [Golgi apparatus] and the endoplasmic reticulum), fragment and disperse toward the periphery of the cell. The nucleolus disappears (disperses) as well, and the centrosomes begin to move to opposite poles of the cell. Microtubules that will form the mitotic spindle extend between the centrosomes, pushing them farther apart as the microtubule fibers lengthen. The sister chromatids begin to coil more tightly with the aid of condensin proteins and now become visible under a light microscope.

Prometaphase (the “first change phase”): Many processes that began in prophase continue to advance. The remnants of the nuclear envelope fragment further, and the mitotic spindle continues to develop as more microtubules assemble and stretch across the length of the former nuclear area. Chromosomes become even more condensed and discrete. Each sister chromatid develops a protein structure called a kinetochore in its centromeric region ((Figure)). The proteins of the kinetochore attract and bind to the mitotic spindle microtubules. As the spindle microtubules extend from the centrosomes, some of these microtubules come into contact with and firmly bind to the kinetochores. Once a mitotic fiber attaches to a chromosome, the chromosome will be oriented until the kinetochores of sister chromatids face the opposite poles. Eventually, all the sister chromatids will be attached via their kinetochores to microtubules from opposing poles. Spindle microtubules that do not engage the chromosomes are called polar microtubules . These microtubules overlap each other midway between the two poles and contribute to cell elongation. Astral microtubules are located near the poles, aid in spindle orientation, and are required for the regulation of mitosis.


Metaphase (the “change phase”): All the chromosomes are aligned in a plane called the metaphase plate , or the equatorial plane, roughly midway between the two poles of the cell. The sister chromatids are still tightly attached to each other by cohesin proteins. At this time, the chromosomes are maximally condensed.

Anaphase (“upward phase”): The cohesin proteins degrade, and the sister chromatids separate at the centromere. Each chromatid, now called a single chromosome, is pulled rapidly toward the centrosome to which its microtubule is attached. The cell becomes visibly elongated (oval shaped) as the polar microtubules slide against each other at the metaphase plate where they overlap.

Telophase (the “distance phase”): the chromosomes reach the opposite poles and begin to decondense (unravel), relaxing once again into a stretched-out chromatin configuration. The mitotic spindles are depolymerized into tubulin monomers that will be used to assemble cytoskeletal components for each daughter cell. Nuclear envelopes form around the chromosomes, and nucleosomes appear within the nuclear area.

Cytokinesis

Cytokinesis , or “cell motion,” is sometimes viewed as the second main stage of the mitotic phase, during which cell division is completed via the physical separation of the cytoplasmic components into two daughter cells However, as we have seen earlier, cytokinesis can also be viewed as a separate phase, which may or may not take place following mitosis. If cytokinesis does take place, cell division is not complete until the cell components have been apportioned and completely separated into the two daughter cells. Although the stages of mitosis are similar for most eukaryotes, the process of cytokinesis is quite different for eukaryotes that have cell walls, such as plant cells.

In animal cells, cytokinesis typically starts during late anaphase. A contractile ring composed of actin filaments forms just inside the plasma membrane at the former metaphase plate. The actin filaments pull the equator of the cell inward, forming a fissure. This fissure is called the cleavage furrow . The furrow deepens as the actin ring contracts, and eventually the membrane is cleaved in two ((Figure)).

In plant cells, a new cell wall must form between the daughter cells. During interphase, the Golgi apparatus accumulates enzymes, structural proteins, and glucose molecules prior to breaking into vesicles and dispersing throughout the dividing cell. During telophase, these Golgi vesicles are transported on microtubules to form a phragmoplast (a vesicular structure) at the metaphase plate. There, the vesicles fuse and coalesce from the center toward the cell walls this structure is called a cell plate . As more vesicles fuse, the cell plate enlarges until it merges with the cell walls at the periphery of the cell. Enzymes use the glucose that has accumulated between the membrane layers to build a new cell wall. The Golgi membranes become parts of the plasma membrane on either side of the new cell wall ((Figure)).


G0 Phase

Not all cells adhere to the classic cell-cycle pattern in which a newly formed daughter cell immediately enters the preparatory phases of interphase, closely followed by the mitotic phase, and cytokinesis. Cells in G0 phase are not actively preparing to divide. The cell is in a quiescent (inactive) stage that occurs when cells exit the cell cycle. Some cells enter G0 temporarily due to environmental conditions such as availability of nutrients, or stimulation by growth factors. The cell will remain in this phase until conditions improve or until an external signal triggers the onset of G1. Other cells that never or rarely divide, such as mature cardiac muscle and nerve cells, remain in G0 permanently.

Which of the following is the correct order of events in mitosis?

  1. Sister chromatids line up at the metaphase plate. The kinetochore becomes attached to the mitotic spindle. The nucleus reforms and the cell divides. Cohesin proteins break down and the sister chromatids separate.
  2. The kinetochore becomes attached to the mitotic spindle. Cohesin proteins break down and the sister chromatids separate. Sister chromatids line up at the metaphase plate. The nucleus reforms and the cell divides.
  3. The kinetochore becomes attached to the cohesin proteins. Sister chromatids line up at the metaphase plate. The kinetochore breaks down and the sister chromatids separate. The nucleus reforms and the cell divides.
  4. The kinetochore becomes attached to the mitotic spindle. Sister chromatids line up at the metaphase plate. Cohesin proteins break down and the sister chromatids separate. The nucleus reforms and the cell divides.

Determine the Time Spent in Cell-Cycle Stages

Problem: How long does a cell spend in interphase compared to each stage of mitosis?

Background: A prepared microscope slide of whitefish blastula cross-sections will show cells arrested in various stages of the cell cycle. (Note: It is not visually possible to separate the stages of interphase from each other, but the mitotic stages are readily identifiable.) If 100 cells are examined, the number of cells in each identifiable cell-cycle stage will give an estimate of the time it takes for the cell to complete that stage.

Problem Statement: Given the events included in all of interphase and those that take place in each stage of mitosis, estimate the length of each stage based on a 24-hour cell cycle. Before proceeding, state your hypothesis.

Test your hypothesis: Test your hypothesis by doing the following:

  1. Place a fixed and stained microscope slide of whitefish blastula cross-sections under the scanning objective of a light microscope.
  2. Locate and focus on one of the sections using the low-power objective of your microscope. Notice that the section is a circle composed of dozens of closely packed individual cells.
  3. Switch to the medium-power objective and refocus. With this objective, individual cells are clearly visible, but the chromosomes will still be very small.

Switch to the high-power objective and slowly move the slide left to right, and up and down to view all the cells in the section ((Figure)). As you scan, you will notice that most of the cells are not undergoing mitosis but are in the interphase period of the cell cycle.



Record your observations: Make a table similar to (Figure) within which to record your observations.

Results of Cell Stage Identification
Phase or Stage Individual Totals Group Totals Percent
Interphase
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
Totals 100 100 100 percent

Analyze your data/report your results: To find the length of time whitefish blastula cells spend in each stage, multiply the percent (recorded as a decimal) by 24 hours. Make a table similar to (Figure) to illustrate your data.

Estimate of Cell Stage Length
Phase or Stage Percent Time in Hours
Interphase
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis

Draw a conclusion: Did your results support your estimated times? Were any of the outcomes unexpected? If so, discuss those events in that stage that may have contributed to the calculated time.

Section Summary

The cell cycle is an orderly sequence of events. Cells on the path to cell division proceed through a series of precisely timed and carefully regulated stages. In eukaryotes, the cell cycle consists of a long preparatory period, called interphase, during which chromosomes are replicated. Interphase is divided into G1, S, and G2 phases. The mitotic phase begins with karyokinesis (mitosis), which consists of five stages: prophase, prometaphase, metaphase, anaphase, and telophase. The final stage of the cell division process, and sometimes viewed as the final stage of the mitotic phase, is cytokinesis, during which the cytoplasmic components of the daughter cells are separated either by an actin ring (animal cells) or by cell plate formation (plant cells).

Art Connections

(Figure) Which of the following is the correct order of events in mitosis?

  1. Sister chromatids line up at the metaphase plate. The kinetochore becomes attached to the mitotic spindle. The nucleus reforms and the cell divides. Cohesin proteins break down and the sister chromatids separate.
  2. The kinetochore becomes attached to the mitotic spindle. Cohesin proteins break down and the sister chromatids separate. Sister chromatids line up at the metaphase plate. The nucleus reforms and the cell divides.
  3. The kinetochore becomes attached to the cohesin proteins. Sister chromatids line up at the metaphase plate. The kinetochore breaks down and the sister chromatids separate. The nucleus reforms and the cell divides.
  4. The kinetochore becomes attached to the mitotic spindle. Sister chromatids line up at the metaphase plate. Cohesin proteins break down and the sister chromatids separate. The nucleus reforms and the cell divides.

(Figure) D. The kinetochore becomes attached to the mitotic spindle. Sister chromatids line up at the metaphase plate. Cohesin proteins break down and the sister chromatids separate. The nucleus reforms and the cell divides.

Free Response

Briefly describe the events that occur in each phase of interphase.

During G1, the cell increases in size, the genomic DNA is assessed for damage, and the cell stockpiles energy reserves and the components to synthesize DNA. During the S phase, the chromosomes, the centrosomes, and the centrioles (animal cells) duplicate. During the G2 phase, the cell recovers from the S phase, continues to grow, duplicates some organelles, and dismantles other organelles.

Chemotherapy drugs such as vincristine (derived from Madagascar periwinkle plants) and colchicine (derived from autumn crocus plants) disrupt mitosis by binding to tubulin (the subunit of microtubules) and interfering with microtubule assembly and disassembly. Exactly what mitotic structure is targeted by these drugs and what effect would that have on cell division?

The mitotic spindle is formed of microtubules. Microtubules are polymers of the protein tubulin therefore, it is the mitotic spindle that is disrupted by these drugs. Without a functional mitotic spindle, the chromosomes will not be sorted or separated during mitosis. The cell will arrest in mitosis and die.

Describe the similarities and differences between the cytokinesis mechanisms found in animal cells versus those in plant cells.

There are very few similarities between animal cell and plant cell cytokinesis. In animal cells, a ring of actin fibers is formed around the periphery of the cell at the former metaphase plate (cleavage furrow). The actin ring contracts inward, pulling the plasma membrane toward the center of the cell until the cell is pinched in two. In plant cells, a new cell wall must be formed between the daughter cells. Due to the rigid cell walls of the parent cell, contraction of the middle of the cell is not possible. Instead, a phragmoplast first forms. Subsequently, a cell plate is formed in the center of the cell at the former metaphase plate. The cell plate is formed from Golgi vesicles that contain enzymes, proteins, and glucose. The vesicles fuse and the enzymes build a new cell wall from the proteins and glucose. The cell plate grows toward and eventually fuses with the cell wall of the parent cell.

List some reasons why a cell that has just completed cytokinesis might enter the G0 phase instead of the G1 phase.

Many cells temporarily enter G0 until they reach maturity. Some cells are only triggered to enter G1 when the organism needs to increase that particular cell type. Some cells only reproduce following an injury to the tissue. Some cells never divide once they reach maturity.

What cell-cycle events will be affected in a cell that produces mutated (non-functional) cohesin protein?

If cohesin is not functional, chromosomes are not packaged after DNA replication in the S phase of interphase. It is likely that the proteins of the centromeric region, such as the kinetochore, would not form. Even if the mitotic spindle fibers could attach to the chromatids without packing, the chromosomes would not be sorted or separated during mitosis.

Glossary


DNA content doubling in interphase - Biology

After M phase (discussed below), the daughter cells each begin a new cycle by proceeding to interphase. Each stage of interphase has a distinct set of specialized biochemical processes that prepares the cell for initiation of cell division (see figure below).

Interphase begins with G1 (G stands for gap) phase. During this phase, the cell makes a variety of proteins that are needed for DNA replication.

During S phase, which follows G1 phase, all of the chromosomes are replicated. Following replication, each chromosome now consists of two sister chromatids (see figure below). Thus, the amount of DNA in the cell has effectively doubled, even though the ploidy, or chromosome count, of the cell remains at 2n. Note: Chromosomes double their number of chromatids post replication but the nuclei remains diploid as the number of centromeres and chromosomes remains unchanged. Hence, the number of chromosomes in the nucleus, which determines the ploidy, remains unchanged from the beginning to the end of the S phase.

Following S phase, the cell enters G2 phase. During G2, the cell synthesizes a variety of proteins. Of particular significance to the cell cycle, most microtubules &ndash proteins that are required during mitosis &ndash are produced during G2.

Not all cells are continually replicated. Non-replicating cells are found in a stage of the cell cycle called G0. These cells may be quiescent (dormant) or senescent (aging or deteriorating). Such cells generally enter the G0 phase from G1. Cells may remain quiescent in G0 for an indeterminate period of time (when no more new cells are needed), only to re-enter G1 phase and begin dividing again under specific conditions. While quiescent cells may re-enter the cell cycle, senescent cells do not. One reason that cells trigger senescence is to ensure that damaged or defective DNA sequences is not passed on to daughter cells.

Checkpoints

Cell cycle progression requires a sequence of processes, with later events dependent on the completion of earlier ones. This dependency ensures that each cell division accurately replicates the genome and transmits it to daughter cells. Checkpoints control the cell&rsquos progress through the cell cycle, and ensure that key processes such as DNA replication and DNA damage repair are completed before the cell cycle is allowed to progress into the next stage. Checkpoints also ensure that both daughter cells receive the same number of chromosomes and that daughter cells are genetically identical to the parents.


Campbell Biology: Ninth Edition - Chapter 12: The Cell Cycle Flashcards

Chapter 12
Cell Division / Mitosis
Vocabulary: gene, cell division, chromosomes, somatic cells, gametes, chromatin, sister chromatids, centromere, mitosis, cytokinesis, meiosis, mitotic phase, interphase, centrosome, aster, kinetochore, cleavage furrow, cell plate, mitotic spindle, binary fission, transformation, benign tumor, malignant tumor, metastasis
Objectives:
After attending lectures and studying the chapter, the student should be able to:
1. Define gene as it relates to the genetic material in a cell.
2. Describe the composition of the genetic material in bacteria, in archaea, and in eukaryotic cells.
3. State the location of the genetic material in prokaryotic and eukaryotic cells.
4. Distinguish between the structure of the genetic material as chromatin and as
chromosomes.
5. Distinguish between the function of the genetic material as chromatin and as
chromosomes.
6. Relating to eukaryotic cells:
a. Describe the centromere region in the genetic material.
b. State the role of cohesins in duplicated genetic material.
c. Describe the sister chromatids of a duplicated chromosome.
d. State the role of the kinetochores on the chromatids at the centromere of a duplicated
chromosome.
e. Describe spindle fibers and state their role in the separation of chromosomes during eukaryotic cell division.
f. Describe the role of centrosomes in the formation of the spindle apparatus.
g. Distinguish between a gene and an allele.
h. Describe homologous chromosomes.
i. Distinguish between an individual's genome and karyotype.
j. State the number of chromosomes in human haploid cells and in human diploid cells.
k. State which cells in humans are haploid, which cells are diploid, and which cells are neither.
7. State the two major parts of the cell cycle.
8. Describe the differences of growth characteristics between a cancerous (transformed) cell and a normal cell.
8. Relating to the prokaryotic cell cycle:
a. State the number of chromosomes in a prokaryotic cell.
b. State the cellular activities that occur during interphase.
c. Show the process of binary fission that is prokaryotic cell division.
9. Relating to the eukaryotic cell cycle:
a. Distinguish between interphase and cell division.
b. Distinguish between the G1, S, and G2 phases of interphase.
c. Define karyokinesis and cytokinesis.
d. State the two types of karyokinesis.
e. Distinguish between the M and C phases of cell division.
f. State when in the cell cycle duplication of the genetic material occurs.
10. Relating to cell division involving mitosis (mitosis + cytokinesis):
a. Define mitosis.
b. Explain why mitosis is sometimes considered "duplication division".
c. State what 1 human diploid cell becomes after mitosis plus cytokinesis.
d. State the reason humans undergo cell division involving mitosis.
e. State which cells in humans undergo cell division involving mitosis.
f. Be able to describe, draw, and recognize the 4 stages of mitosis.
g. Describe the cleavage-furrow process of cytokinesis in animal cells.
h. Describe the cell-plate process of cytokinesis in plant cells.