Week 10 Lab 10 Mitosis and Meiosis

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Note: All your answers to questions must be in Red or other color (not including blue) for easier grading. Points will be deducted if you do not distinguish your answers. Lab 10. Mitosis and Meiosis Objectives:  List the stages of the cell cycle in order.  Describe the cellular events that occur during interphase (be sure to include the specific portions of interphase such as G1, S and G2).  Describe the overall process of mitosis (what is the purpose and the outcome of mitosis).  Describe the events of prophase of mitosis.  Describe the events of metaphase of mitosis.  Describe the events of anaphase of mitosis.  Describe the events of telophase of mitosis. Vocabulary:  Daughter Cells  Interphase  Prophase  Metaphase  Anaphase  Telophase  Cytokinesis  Cell theory  Mitosis  Cell cycle  Checkpoints Introduction: The cell cycle is like the life cycle but for cells. It is essentially a series of events involving self-growth and the cell division that essentially produces our 2 daughter cells. Cell cycle composed of Interface, Meiotic Phase, & Cytokinesis. In interface, G1, the cells are growing and undergo protein synthesis, after G1, the cells decide to make a copy of themselves and create a new cell (a clone) through mitosis, we are going to duplicate the DNA and that is what happens in the S phase but also replication of the centrosome. In G2, it involves further growth and protein synthesis, also involves organelles replicating as the new cell is created. After G2, the cell is ready to undergo mitosis, that is the meiotic phase. According to the cell theory of biology, all cells arise from pre-existing cells. Most eukaryotes accomplish nuclear division by either mitosis or meiosis, which by the most part involves the separate but often coordinated

processes of cytokinesis (division of the cytoplasm and organelles). Mitosis without the co-occurrence of cytokinesis leads to the production of multinucleated cells, such as those found naturally in skeletal muscle and cardiac muscle. Mitosis produces two diploid (2n) somatic cells that are genetically identical to each other and the original parent cell , whereas meiosis produces four haploid (n) gametes that are genetically unique from each other and the original parent (germ) cell . Mitosis is commonly divided into four major phases: prophase , metaphase , anaphase , and telophase . You may find that some accounts of mitosis further subdivide the process to include prometaphase between prophase and metaphase. In this exercise, we will consider prometaphase a component of prophase. Figure 1A. The Cell Cycle. A cell moves through a series of phases in an orderly manner. During interphase, G1 involves cell growth and protein synthesis, the S phase involves DNA replication and the replication of the centrosome, and G2 involves further growth and protein synthesis. The mitotic phase follows interphase. Mitosis is nuclear division during which duplicated chromosomes are segregated and distributed into daughter nuclei. Usually, the cell will divide after mitosis in a process called cytokinesis in which the cytoplasm is divided, and two daughter cells are formed. Regulation at Internal Checkpoints It is essential that daughter cells be exact duplicates of the parent cell. Mistakes in the duplication or distribution of the chromosomes lead to

mutations that may be passed forward to every new cell produced from the abnormal cell. To prevent a compromised cell from continuing to divide, there are internal control mechanisms that operate at three main cell cycle checkpoints at which the cell cycle can be stopped until conditions are favorable. These checkpoints occur near the end of G 1 , at the G 2 –M transition, and during metaphase ( Figure 1B ). Figure 1B: The cell cycle is controlled at three checkpoints. Integrity of the DNA is assessed at the G1 checkpoint. Proper chromosome duplication is assessed at the G2 checkpoint. Attachment of each kinetochore to a spindle fiber is assessed at the M checkpoint. Interphase Cells spend most of their time in a stage called interphase. During this phase, the nuclear envelope surrounds the nucleus. There may be one or more nucleoli (dark, condensed regions) visible within the nucleus. The material around the nucleoli, contained within the nuclear envelope is DNA in the form of chromatin. This will not pick up a stain well and so will not appear as distinct shapes within the nucleus. Find these indicators of interphase in Figure 2A in the image below.

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2A 2B Figure 2. Cells in an onion root in 2A interphase and prophase. Cell A has a large, dark nucleolus surrounded by greyish material (chromatin) that is enclosed within the nuclear membrane. A cell wall makes a box around each cell and the plasma membrane would be located just inside this box, though we cannot easily see it. In cell B, the chromatin is condensing and begins to look like dark, thick strands. It is still contained in the center of the cell, as the nuclear envelope has not finished dissolving. In 2B we see interphase in a whitefish blastula. In contrast, when a cell begins the process of division, the chromatin condenses into visible chromosomes that will pick up a stain and look like dark strings within the nuclear envelope, as seen in cell B in the image above. In order to begin dividing, the cell needs to go through several processes that take place during interphase, including replicating the DNA (occurs in S- phase) and all of the cell contents. Mitosis

Mitosis is the process of dividing the nucleus. To see cells in mitosis, we look in areas of a plant that would be actively growing. These areas where cells are actively dividing are called meristems, such as the root apical meristem. We will also look at a prepared slide of whitefish blastula undergoing mitosis. The blastula is an early embryonic stage in the development of animal cells. Figure 3. A long section of an onion root tip. The edge of the growing tip has been outlined, attempting to show the location of the protoderm cells (located approximately on the black line) vs. the root cap, (think of a hard hat that protects delicate cells; delicate cells are formed in the zone of cell division) which would be located to the exterior of the black line. The root apical meristem is indicated by an arrow that points to the center of the U-shaped line. All cells in the root are derived initially from the root apical meristem. Prophase

4A 4B Figure 4: In 4A Cell A is in late prophase. The chromatin has condensed into visible chromosomes that form dark strands and the nucleolus is no longer visible. In cell B, the chromatin is condensing and some chromosomes are visible, but the nucleolus has not yet dissolved. In cell C, the nuclear envelope is gone, no nucleoli are visible, and the chromosomes are separating from each other. In cell D, the nuclear envelope and nucleoli are distinct, and the chromatin has not yet condensed. In 4B we observe prophase in whitefish blastula cell. Metaphase (including Prometaphase)

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5A 5B Figure 5. In 5A , cell A is in prometaphase. The chromosomes are no longer contained within a nuclear envelope. The spindle fibers are attaching to the kinetochores, but the chromosomes have not yet been pulled into a line. Cell B is in metaphase and the chromosomes are loosely aligned on the metaphase plate. In 5B , we see a whitefish blastula cell undergoing metaphase. The spindle apparatus, centriole, and chromosomes aligned the metaphase plate are visible at this stage. Anaphase

6A 6B Figure 6: In 6A , cell A is in anaphase. The sister chromatids have been pulled apart and now there are two distinct groups of chromosomes on either side of the cell. In 6B , whitefish blastula cell is in anaphase. Telophase 7A

7B Figure 7: In 7A, cell C is in telophase. There are two dark regions where the chromosomes are clustered and decondensing, becoming indistinguishable from each other. A fuzzy line is forming between them, indicated by a black arrow, showing cytokinesis is happening as a new cell wall forms. Cell A is the same cell that is shown in Figure 6. In cell B and D, the chromosomes are grouped together in the center of the cell, though they appear much more orderly in cell D. In cells E and F, the chromosomes are in two distinct groups on either side of the cell but are still distinguishable as individual strands. In the two cells indicated by G, there are distinguishable nucleoli and a clear nuclear envelope. In 7B , we see a whitefish blastula cell undergoing telophase. The arrows indicate a contractile ring forming around the midpoint of the cell. Cytokinesis 8A 8B

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Figure 8: In 8A , Cell D is in telophase. Cytokinesis is not part of mitosis, but happens concurrently with telophase. There are two dark regions where the chromosomes are clustered and decondensing, becoming indistinguishable from each other. A fuzzy line is forming between them, indicated by a white arrow, showing cytokinesis is happening as a new cell wall forms. Cell A has distinguishable chromosomes and a nucleolus. Cell B has partially condensed chromosomes, two nucleoli, and a clear nuclear envelope. Cell C has two groups of chromosomes being pulled to opposite sides of the cell. In 8B , we see a whitefish blastula cell with a contractile ring. This contractile ring is composed of microfilaments of actin and myosin. As this ring contracts, it creates a cleavage furrow that drags the plasma membrane inward, eventually pinching the cell into two new cells. Meiosis Meiosis is more complex than mitosis and involves two nuclear divisions called Meiosis I and Meiosis II . These divisions result in the production of four haploid gametes and allow for genetic variation due to crossing over of genetic material. Prior to the process, interphase involves replication of the DNA. During prophase I , the first meiotic stage, homologous chromosomes move together to form a tetrad and synapsis begins. This is the stage in which crossing over occurs, resulting in the recombination of genes. In metaphase I , the tetrads move to the metaphase plate in the middle of the cell as in mitotic metaphase. Anaphase I brings the tetrads back to their original two-stranded form and moves them to opposite poles. During telophase I , the cell prepares for a second division. In Meiosis II, in prophase II , centrioles move to opposite ends of the chromosome group. In metaphase II , the chromosomes are positioned at the center of each daughter cell. Anaphase II involves the centromere and separation of chromatids. Telophase II occurs when the divided chromosomes separate into different cells, known as haploid cells. Figure 9: Production of gamete (haploid) cells through the process of meiosis I and meiosis II. Gametogenesis (Spermatogenesis and Oogenesis) Gametogenesis, the production of sperm and eggs, takes place through the process of meiosis. During meiosis, two cell divisions separate the paired

chromosomes in the nucleus and then separate the chromatids that were made during an earlier stage of the cell’s life cycle, resulting in gametes that each contain half the number of chromosomes as the parent. The production of sperm is called spermatogenesis and the production of eggs is called oogenesis. Oogenesis Oogenesis occurs in the outermost layers of the ovaries. Similar to spermatogenesis, oogenesis involves the formation of haploid cells from an original diploid cell, the primary oocyte , through meiosis. The female ovaries contain primary oocytes. There are two major differences between the male and female production of gametes. First, oogenesis only leads to the production of one ovum, or egg cell, from each primary oocyte (this is in contrast to the four sperm that are generated from every spermatogonium). Of the four daughter cells that are produced when the primary oocyte divides, three are much smaller than the fourth. These three smaller cells, called polar bodies , eventually disintegrate to leave only the last ovum, secondary oocyte , as the final product of oogenesis. The production of one egg cell via oogenesis normally occurs only once a month, from puberty to menopause.

Figure 10: The process of oogenesis occurs in the ovary’s outermost layer. A primary oocyte begins the first meiotic division, but then arrests until later in life when it will finish this division in a developing follicle. This results in a secondary oocyte, which will complete meiosis if it is fertilized. Spermatogenesis The male testes have tiny tubules containing diploid cells called spermatogonia that mature to become sperm. The basic function of spermatogenesis is to transform each diploid spermatogonium into four haploid sperm cells. This quadrupling is accomplished through meiotic cell division as detailed in the last section. During interphase, before Meiosis I, each spermatogonium’s 46 single chromosomes are replicated to form 46 pairs of sister chromatids. Sister chromatids exchange genetic material through synapsis before the first meiotic division. In Meiosis II, the two daughter cells go through a second division to yield four cells containing a unique set of 23 single chromosomes that, ultimately, mature into four

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sperm cells. Starting at puberty, a male will produce literally millions of sperm every single day for the rest of his life. Figure 11: During spermatogenesis, four sperm result from each primary spermatocyte, which divides into two haploid secondary spermatocytes; these cells will go through a second meiotic division to produce four spermatids.

Human Life Cycle Nearly all animals employ a diploid-dominant life-cycle strategy in which the only haploid cells produced by the organism are the gametes. The gametes are produced from diploid germ cells, a special cell line that only produces gametes. Once the haploid gametes are formed, they lose the ability to divide again. There is no multicellular haploid life stage. Fertilization occurs with the fusion of two gametes, usually from different individuals, restoring the diploid state (Figure 12). Figure 12: In animals, sexually reproducing adults form haploid gametes from diploid germ cells. Part 1: Microscopic Mitosis In this part of the lab, you will examine 2 different slides:  A cross section of an onion root tip, where cell growth (and consequently mitosis) happens at a rapid rate.  Blastula of a whitefish. The blastula is a distinct stage during embryonic development when a fertilized egg forms a hollow ball of cells. During embryonic development, cells are dividing quickly, and we are more likely to be able to see the varying stages of mitosis. Materials:  Allium slide (the genus of onions)  Whitefish blastula slide  Microscope Procedure:

You must have your own microscope for this lab!  Using correct microscope procedure, observe an onion root tip under high power (40X).  Locate the region of active cell division, known as the root apical meristem, which is about 1 mm behind the actual tip of the root.  Identify and draw a cell in each of the four stages of mitosis in the onion slide. Then draw cells in cytokinesis as well.  Observe the prepared slide of a whitefish blastula under high power (40X).  Identify and draw a cell in each of the four stages of mitosis in the whitefish blastula slide. Then draw cells in cytokinesis as well. Results: 1. Draw an onion cell in Prophase. Describe the main points that are occurring in this stage. During the prophase of mitosis, chromosome condensation occurs, the formation of the spindle apparatus begins, the nuclear envelope (nuclear membrane) breaks down, and the centrosome movement plays a pivotal role in preparing for cell division. 2. Draw an onion cell in Metaphase. Describe the main points that are occurring in this stage. During metaphase, several key events occur in the onion cell, starting with chromosome alignment, (which went through condensation during prophase), the spindle fibers have fully formed by metaphase. They help position and hold the chromosomes at the metaphase plate, that is the imaginary plane in the middle of the cell nucleus where the chromosomes align.

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3. Draw an onion cell in Anaphase. Describe the main points that are occurring in this stage. During this phase, the spindle fibers play a key role in this process by exerting tension and pulling the sister chromatids apart and move toward the opposite poles of the cell. [ This phase I had a difficult time finding one. The 3 rd picture I included is to explain that I know what it looks like, and I tried as closest to find one that resembles it, so I listed 2 different pictures]. 4. Draw an onion cell in Telophase. Describe the main points that are occurring in this stage. In Telophase, the main point is to ensure the proper distribution of genetic material into daughter cells and the reconstitution of functional nuclei, also the nucleolus, to prepare for interphase. In short cell division begins.

5. Draw an onion cell in Interphase. Describe the main points that are occurring in this stage. Interphase allows the cell to grow, carry out its normal functions, and duplicate its DNA before it is ready to divide, it is an influential time of the cell cycle. 6. Draw an onion cell in Cytokinesis. Describe the main points that are occurring in this stage. The main point of cytokinesis is the physical division of the cell, which ensures that each daughter cell receives a complete set of chromosomes as a new cell wall forms as seen by the line that is being established, allowing them to function as independent units. 7. Draw a whitefish blastula cell in Prophase.

8. Draw a whitefish blastula cell in Metaphase. 9. Draw a whitefish blastula cell in Anaphase. 10. Draw a whitefish blastula cell in Telophase. 11. Draw a whitefish blastula cell in Interphase.

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Part 2: Estimating Relative Time Spent in Each Stage of Mitosis If you froze time and took a snapshot of a group of cells in a living organism, you could estimate the relative amount of time a cell spends in each stage of the cell cycle simply by counting the number of cells in each stage. For example, if there are 100 cells in your view and 90 of them are in prophase, you can assume that the cells spend most of the time in prophase. Cells in your body reproduce at different rates. Skin cells reproduce frequently (about once per day); liver cells reproduce rarely (about once per year). Some specialized cells like nerve and muscle cells almost never reproduce and are in a special stage called G 0 . The whole process of mitosis, prophase to telophase, takes approximately 90 min. In plants, an area of rapid growth is the tips of roots. This exercise uses onion root tips to illustrate the amount of time spent in each phase of mitosis. Materials:  Allium slide  Microscope Procedure:  Return to the slide of the onion root tip. Using correct microscope procedure, observe an onion root tip under high power (40X).  Choose ONE view and then carefully COUNT 36 cells identify how many are in each stage of the cell cycle. o Online classes can use this link instead and do the interactive activity with preselected images:  http://www.biology.arizona.edu/cell_bio/activities/cell_cycle/ cell_cycle.html Results: Interpha se Prophas e Metapha se Anaphas e Telophas e Total Number of Cells 20 10 3 2 1 36 Percentag e of Cells 55.56 27.78 8.33 5.56 2.77 100% Conclusion: 12. Based on your observations, what can you conclude from this experiment regarding the percent of time? In preparation for mitosis, the onion cells spend most of their time in interphase, and the second most amount of time in prophase. As for the remaining phases, it takes

a while for metaphase, to align, and anaphase to separate, whereas in telophase it happens fast. Additional Questions: 13. In which phase does DNA replication take place? DNA replication occurs during the S phase of interphase. This process is essential for providing each new daughter cell with a full complement of genetic material. 14. What is the cell doing during G 1 and G 2 ? G 1 is the first growth phase, G 2 is the second growth phase. In G 1 , the cell grows and carries out metabolic functions and in G 2 the cell continues to grow, synthesize proteins, and prepare for cell division (mitosis). 15. What are two examples of cells that stay in G 0 permanently? Two examples are nerve and heart muscle cells that typically enter the G 0 phase and remain there for the duration of their life since they do not undergo cell division once they reach maturity. 16. What happens during G 1 Checkpoint? It is a critical decision point in the cell cycle, at which cells assess various factors (cell size, nutrients, growth) to determine whether to proceed with cell division. Once the cell passes the G 1 checkpoint, enters S phase, it is permanently committed to division. 17. What happens during G 2 Checkpoint? It ensures that all chromosomes have been completely and accurately replicated during the S phase. Including checking for any DNA damage/errors in replication. 18. What is mitosis? Mitosis is the division of the nucleus (nuclear division). A process of cell division in which a single parent cell divides to produce two identical daughter cells. 19. In order, what are the four main stages of mitosis? The four main stages are prophase, metaphase, anaphase, and telophase. 20. What structure is involved in moving chromosomes during mitosis? Spindle fibers (composed of individual microtubule fibers) are responsible for separating the duplicated chromosomes (sister chromatids) and moving them to opposite poles of the cell, ensuring that each daughter cell receives a complete set of chromosomes.

21. At the completion of mitosis when the cell divides, what name is given to the two new cells? The name given is daughter cells (identical). 22. What are the 3 stages of interphase? G1 phase aka cell growth or Got 1 phase, S phase or DNA synthesis, and G2 phase which is also cell growth. 23. Why did we use the onion root tip to view mitosis? The onion root tip contains regions with actively dividing cells and their rapid division makes it an excellent choice for studying mitosis. 24. Compare and contrast mitosis of animal and plant cells. Animal cells: cytokinesis involves the formation of a structure called the cleavage furrow. Plant cells: the process of cytokinesis is a bit different due to the presence of a rigid cell wall surrounding the plant cell membrane; therefore, instead of cleavage furrow, a structure called the “cell plate” forms. These differences in cytokinesis are unique structural features of animal and plant cells in that animal cells are more flexible and plant cells have the rigidity of a cell wall to contend with. However, both reach the same goal of dividing the cell into two daughter cells. Part 3: Microscopic Meiosis In flowering plants such as Lilium , meiosis occurs in the reproductive organs of the flower. Pollen (sperm) is produced in the anthers, and ovules (eggs) are produced in the pistil. Inside the anther, thousands of diploid cells called meiocytes undergo meiosis to produce spherical tetrads, each containing 4 haploid cells. These haploid cells will then mature into pollen grains. In today’s experiment you will be using a prepared slide of a lily anther where cells are undergoing meiosis. Materials:  Lily anther slide  Microscope Procedure:  Observe the prepared slide of a lily anther under high power (40X).  Identify and draw a cell in each of the four stages of meiosis II in the lily anther slide. Results:

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25. Draw a lily anther cell in Prophase I. Describe the main points that are occurring in this stage. Nuclear membrane breaks down, chromatin condenses, spindle forms and attaches to kinetochores. 26. Draw a lily anther cell in Metaphase I. Describe the main points that are occurring in this stage. Microtubules align homologous chromosome pairs metaphase plate. 27. Draw a lily anther cell in Metaphase II. Describe the main points that are occurring in this stage. Microtubules align chromosomes along metaphase plate. 28. Draw a lily anther cell in Anaphase II. Describe the main points that are occurring in this stage. Kinetochore microtubules shorten, pulling sister chromatids to opposite poles, polar microtubules elongate, lengthening dividing cell.

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29. Draw a lily anther cell in Telophase II. Describe the main points that are occurring in this stage. Nuclear membrane reforms, and chromatin decondenses, and cell plate begins to form. Part 4: Modeling Meiosis Variation The gametes produced in meiosis aren’t genetically identical to the starting cell, and they also aren’t identical to one another. This variation is obtained by two events: crossing over and random orientation of homologue pairs. In this experiment you are going to model the process of meiosis. Materials:  Coralina modeling beads  11 pieces of white paper  Pencil/pen Procedure:  Set up half of the beads exactly as follows, representing genes on the chromosome of a hypothetical critter. We will assume that the critter is diploid (2n) and has three different chromosomes. Because the critter has two copies of each of the three chromosomes, the diploid number is 6 (2 × 3 = 6).

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 This is what your critter’s chromosomes look like in the unreplicated form. Note that there are six chromosomes here consisting of three homologous pairs. Each chromosome pair consists of a maternal and paternal version of the chromosome. The maternal and paternal versions are represented by the respective bead color.  Replicate your chromosomes! Make enough copies of each chromosome to represent both paternal and maternal chromosomes in a replicated form, as shown below. Note that the sister chromatids are identical in color. Be sure you can identify the sister chromatids, chromosomes, and the difference between a replicated and non-replicated form.

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 Using your maternal and paternal sets of replicated chromosomes and this lab (or the text) as a reference, practice the process of meiosis until you are very comfortable with it. Each person in the group should practice the entire process.  Don’t forget crossing over when you simulate prophase I.  This time, demonstrate the principle of independent assortment by determining how many different gametes you can form with three homologous pairs.  Use the chromosomes to demonstrate all the different ways they can line up on the metaphase plate. Conclusion: 30. How many possible gametes can be formed following meiosis (excluding crossing over events) from an original cell that contains a diploid number of six (2 n = 6)? [Note: The number of possible gametes = 2 n where n is the number of chromosomes per set.] 3 possible gametes. 31. How many possible gametes can be formed following meiosis (excluding crossing over events) from an original cell that contains a diploid number of 46 (2 n = 46)? That would be 23. 32. What is it called when homologous chromosomes exchange genetic material during prophase I? Chromosomes can exchange genetic information during a process called crossing over which happens in metaphase I. 33. During metaphase I, how do homologous chromosomes align? The homologous chromosomes align along the cell’s equatorial plane; in the center/middle. 34. Each human sperm and egg should have 23 chromosomes, so fertilization will produce a zygote with 46 chromosomes; this zygote will develop into a healthy embryo with 46 chromosomes in each cell. Licenses and Attributions  " Mitosis and the Cell Cycle " by Lumen Learning , LibreTexts is licensed under CC-4.0-BY .  " Mitosis (Activity) " by Bio-OER , LibreTexts is licensed under CC BY-NC- SA .  " Interphase, Mitosis, and Cytokinesis " by Maria Morrow , LibreTexts is licensed under CC BY-NC .

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 " Mitosis " by Lumen Learning , LibreTexts is licensed under CC-4.0-BY .  " The Cell Cycle " by OpenStax , LibreTexts is licensed under CC BY 4.0 .  " Gametogenesis (Spermatogenesis and Oogenesis) " by LibreTexts is licensed under CC BY-SA 4.0.  " Meiosis " by OpenStax , LibreTexts is licensed under CC BY 4.0 .  " Meiosis and Gametogenesis " by LibreTexts is licensed under CC BY 4.0 .  https://biology4isc.weebly.com/c-cell-cycle-and-cell-division.html  https://www.southalabama.edu/biology/notes/121%20lab/_baks/BLY121 _lab08_Mitosis.pdf.0001.25e3.bak  http://sites.fas.harvard.edu/~bs50/Mit%20Mei%20Notebook.pdf

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Week 10 Lab 10 Mitosis and Meiosis

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