Experiment Mitosis and Meiosis Margaret Vorndam, M.S. Version 42-0094-00-01 Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise. Experiment Summary: Students will have the opportunity to learn how to differentiate between mitosis and meiosis. They will use onion root tip and whitefish blastula slides to view the stages of mitosis and will model these stages using chromosome beads. Students will observe slides of ovaries and testes and will simulate meiosis using chromosome beads. www.LabPaq.com 207 © Hands-On Labs, Inc. Experiment Mitosis and Meiosis Objectives ●● To explain how chromosomes recombine during sexual reproduction ●● To understand how chromosomal sorting differs for meiosis and mitosis ●● To name the various stages of mitosis and meiosis, and recognize cells in various stages of division ●● To understand why crossing-over of genes in chromosomes can be positive or negative in survival of the species Estimated Time Required to Complete the Experiment: 4 to 8 hours total www.LabPaq.com 208 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis materials Materials From: Student Provides Label or Box/ Bag: Qty Item Description: 1 1 1 1 1 1 Scissors Straw, cut into short pieces Flat surface to represent cell Transparent tape Thread Microscope Chromosome Simulation Chromosome Kit Bag Simulation Kit Bag 1 Chromosome Kit-BK - Centromeres 4 Color Bead Set Slide Box BK-2A 1 1 1 1 Slide - Onion Root Tip Slide - Ovary Slide - Testis Slide - Whitefish Blastula Slide Box BK-2A Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq. www.LabPaq.com 209 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Discussion and Review All living organisms grow through division or enlargement of cells. Living organisms also reproduce new organisms through asexual means by fission or mitosis, or through gamete production, followed by fertilization. In this experiment we will examine the processes responsible for organisms’ ability to grow and reproduce. Cell division, also referred to as replication, is accomplished through the process of mitosis (Gk. mitos, thread + -osis). During cellular mitosis, cell chromosomes that contain the genetic code for the organism are replicated; the two chromosome sets are divided, the cell splits, and a set of chromosomes goes to each cell. Since no new genetic material is added during this process (discounting possible crossover mutations that may occur within the dividing cells), this process is referred to as asexual reproduction. Organisms use this process of mitotic cellular replication to increase numbers or replace dead cells in cellular tissues and to repair damage from injuries. Simple organisms may exclusively use mitosis to reproduce exact copies of themselves. An example of this is a hydra, a Cnidarian that produces buds along its body which break away from the parent organism and grow into a new hydra. Other examples of mitotically facilitated reproduction include plant cuttings used to propagate new plants, potato tubers used to grow potatoes, and plants grown from bulbs. Cloning is another example of this type of process. In each example there is no reduction in the number of chromosomes or any exchange of genetic material with another like organism. Each new cell has the same number of chromosomes as did the original cell and is identical to the parent from which it was derived. Since no chromosomal division occurred in these mitotically divided cells, the total number of chromosomes is referred to as the 2n (diploid)1 number, where n equals the number of chromosome pairs in the cell. In contrast, during sexual reproduction cellular replication also occurs, but an exchange of genetic material is necessary.2 In this process both meiosis (Gk. meiosis, diminution), a halving of the organism’s chromosome number, in combination with mitosis results in a recombination of the parent gametes in the new offspring. An organism that manifests traits of each parent is produced, containing genetic information that, in combination, is expressed differently than either parent alone. This exchange can convey an added capacity to the new organism/generation of organisms to respond to environmental conditions. Why? 1 Although the diploid number of chromosomes is the number that we commonly associate with mitosis, the student should be aware that some organisms do exist in polyploidy states, where more than one set of chromosomes are present. Terms such as triploid (3n), tetraploid (4n), etc. are associated with these organisms’ normal sets of chromosomes. 2 Some organisms carry both male and female reproductive parts and are able to self-pollinate. Humans must reproduce by the joining of sperm from a male and the egg from a female. www.LabPaq.com 210 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Mitosis – The Life Cycle of a Cell: Cells are “born” when mitosis causes cells to split, creating two where there was one. Neither resulting cell is truly the originating or parent cell since both retain portions of the genetic material - organelles, cytoplasm, and outer membrane - from the original cell during the split. Like organisms, each cell has a finite longevity that is governed by its genetic material. After a period of existence genes within the cell that cause the cell to die are turned on, a process referred to as apoptosis. Why is this process of cell death necessary, and what benefit might it have to the organism? Until apoptosis occurs, active cells go through life cycles that are akin to the sequence presented in the life cycle figure located in Exercise 1. Figure 1: Reproduction in Potato Plants www.LabPaq.com 211 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Exercise 1: Mitosis in Animal and Plant Cells Search Key Terms: Whitefish blastula, onion root tip, mitosis, plant cell division, animal cell division, centrosome, microtubule Although the outcome of mitosis is the same for plant and animal cells, there are some differences in the process. We will examine the processes in an early embryonic stage of the whitefish embryo, an example of animal cells, and in onion root tips representing plant cells. Both cell types’ chromosomes are large and easy to observe under a microscope. In plant mitosis, plants have the added task of dividing the cell wall to form new cell walls between the two cells. This step occurs during Telophase. Unlike animal cells, dividing plant cells do not form centrioles and asters. In plants, the centrosome is the “microtubule organizing center” in the cell and is responsible for spindle development. All other steps are similar to those outlined for animal cell mitosis. PROCEDURE 1. Observe the prepared slide of the whitefish blastula under the microscope. 2. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase and Telophase. 3. For each drawing, label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cleavage furrow formation during Cytokinesis, and centrioles. It is unlikely that you will find all of these structures. Why? 4. Observe the slide of the onion root tip under the microscope. 5. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase and Telophase. 6. Label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cell wall formation region, and centrioles. It is unlikely that you will find all of these structures. Why? www.LabPaq.com 212 ©Hands-On Labs, Inc. Experiment Mitotic Phase Interphase 80%-100% ~ 18-24 hours Cell Activity Mitosis and Meiosis Chromosome Activity Description of Activity Cell nucleus contains nuclear material. Centrioles are paired outside of the nuclear membrane. Cell organelles replicate in cytosol (cytoplasm). The homologous pairs of chromosomes (one originally from mother-M (Black) and one from father- F (Light Grey) replicate exact copies of themselves called sister chromatids joined to other sister chromatid at centromere. This process happens for B e f o r e A f t e r every cell chromosome, but, we will follow only one I n t e r p h a s e I n t e r p h a s e chromosome 16 pair through the mitotic process. 2n 2n Individual balls of the chromosomes represent gene locations. Cell’s nuclear membrane temporarily dissipates. Nucleolus, if present, disappears. Centrioles move to opposite poles of cell. Chromatid pairs (containing two sister chromatids) shorten and thicken. Prophase 2n The mitotic spindle begins to form in the former region of the nucleus. Microtubules appear in the vicinity of each centriole, forming an aster. The centromere of each pair of chromatids captures a spindle fiber as the fibers build between the opposite centriole poles. At this stage, the number of cell chromosomes can be counted since the chromatid pairs are distinct. Prometaphase 2n Nuclear membrane gone. Centrioles are attached by spindle fibers to centromeres of chromatid pairs, now located along the equator (metaphase plate) of the cell. Metaphase 2n Sister chromatids split at the centromere. Each is drawn to an opposite centriole by the attached spindle fiber. Once at the centriole, like chromatids such as chromosome 16 again join to form homologous chromosome pairs. Each centriole now has a full compliment of chromosomes, as did the original cell at the beginning of Interphase, above. Cytokinesis (cytoplasm division) begins. Anaphase 2n Nuclear membrane forms around homologous chromosome pairs. Nucleolus reappears, if present originally. Chromosomes lengthen and become Telophase threadlike in the nucleus. Cytoplasm cleavage In the cell continues until separation into two distinct, but In the cell nucleus of exact, replicates of the original cell, result. nucleus of one cell – 2n the other cell – 2n Each cell enters Interphase and prepares for its own mitotic episode as the cycle of cell division continues. Go back to top for next cycle. Figure 2: Life Cycle of a Typical Cell www.LabPaq.com 213 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Exercise 2: Meiosis in Animals Meiosis – The Production of Gametes: Sex cells called gametes are produced through meiosis, also called gametogenesis3 (= production of gametes) in animals and plants. Fertilization, the combining of gametes, can result in the formation of an embryo and ultimately, a new organism that shares genetic traits from both parent organisms. In animals, eggs and sperm are the gametes produced as a result of meiosis during sexual reproduction. In animals, gametogenesis occurs in specific organs. In the female, eggs are produced through oogenesis4 in the ovary. In the male, sperm are produced through spermatogenesis10 in the testes. In plants, gametes take the form of pollen grains produced in the anthers that contain the sperm and the egg that is produced in the ovary of a flower. In angiosperms where seeds are borne within a fruit that developed from the flower ovary, the male and female reproductive organs may be on the same plant. Some animals also bear both male and female organs on the same organism as earthworms do. These animals are referred to as hermaphrodites. Because fertilization in both plants and animals results from the combining of the egg and the sperm into a new cell, the number of chromosomes must be halved to n (haploid) during the course of gametogenesis. Meiosis is the process that accomplishes this halving, or splitting of the chromosome pair into n chromosomes, from the typical cellular 2n number of chromosomes. Meiosis occurs in two distinct steps: meiosis I and meiosis II. During meiosis I the primary 2n gamete-to-be cell (whether a spermatocyte or oocyte) divides, and the chromosome number is halved, from 2n to n. Figure 3 provides a diagram of the meiotic process. During meiosis II the two secondary n spermatocytes divide again, producing four n sperm. However, in the case of the two secondary n cells produced in oogenesis, only one becomes an egg. The other cell forms the first polar body. After meiosis II an n egg and two polar bodies result. Although one would have expected three, the other polar body disintegrates or continues to divide to form other polar bodies. If the egg is fertilized, the polar bodies function to retain and build cytoplasm for the egg. It must be stressed, that the egg does not complete meiosis II unless a sperm fertilizes it. If not fertilized, the egg passes out of the body (animals) or eventually disintegrates (plants). Note that there are many species where the egg will develop into an organism having n number of chromosomes. This phenomenon is known as parthenogenesis. Examples are the drones or male workers in colonies of bees, ants and some termites. There are also examples where, after development, organisms may change sex depending on the environment in which they find themselves. However, we will leave discussion of this topic to a genetics course. 3 The terms, meiosis and gametogenesis, are equivalent. We will use them interchangeably throughout this laboratory, so the student should be aware that he terms refer to the same process. 4 Oogenesis and spermatogenesis are similar processes. These names refer to meiosis and gamete production within an ovary and testis, respectively. www.LabPaq.com 214 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis The n egg cell and the n sperm cell join, and the two nuclei fuse, bringing the new cell to the diploid (2n) state of chromosome number. But one-half of the chromosomes came from the sperm, and the other half came from the egg. In humans the egg and sperm each have n = 23 chromosomes, but the new zygote formed by their union has the 2n = 46 chromosomes found in humans. In this exercise, we will examine the gamete producing structures in animals. Although we will not be observing the equivalent process in plants, the steps are essentially the same. Remember that ovaries are the site of oogenesis (egg production) in female animal organisms, while testes produce the sperm via spermatogenesis. www.LabPaq.com 215 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Meiosis I: Homologous chromosomes synapse and separate in sex gonads 2n = chromosome paired with homologue chromosome. n = chromosome not paired with homologue chromosome Meiotic Phase Cell Activity Chromosome Activity Interphase 1 cell 2n Prophase I 1 cell 2n x 2 Metaphase I 1 cell 2n x 2 Description of Activity Cell nucleus contains nuclear material. A centriole replicates, paired outside. The cell organelles replicate in cytoplasm. A homologue chromosome pair joined by centromere. The nuclear membrane and nucleolus, if present, disappears. A spindle forms from centrosome as centrioles move to cell poles. Synapsis occurs so that each original chromosome homologue is doubled. This results in the chromosome pair attached together, but containing four chromatids. A chromosome pair is referred to as “bivalent” as each now is doubled. All cellular chromosomes (bivalent forms) line up randomly on metaphase plate. There is a 50/50 chance that the future daughter cells will receive either inherited male or female parent chromatid (Chromosomes 16 and 5 are shown for demonstration purposes). Spindle fibers are captured by centromeres of bivalent chromosomes. Homologous chromosome pair separates and move to poles, pulled by spindle fibers. Note in this example that Female parent chromatid pair 5 at one centriole has joined Male parent chromatid pair 16, and that Male parent chromatid pair 5 at other centriole has joined that Female parent chromatid pair 16. Anaphase I 1 cell 2n x 2 Nuclear membrane, nucleolus reappears. Spindle disappears. Cell separates into two cells, each containing a pair of chromatids. However, each pair of chromatids is identical. Telophase I 2 cells n each At end of Telophase I 2 cells n each Figure 3: Meiosis: The Production of Gametes in Preparation for Sexual Reproduction www.LabPaq.com 216 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Meiosis II: Chromatids separate Meiotic Phase Cell Activity Chromosome Activity Description of Activity Prophase II In each cell resulting from Meiosis I: Centriole 2 cells, each replicates. The nuclear going through membrane and nucleolus, meiosis II if present, disappear. n each A spindle forms as the x2 Cell 2 centrioles move to cell Cell 1 poles. Metaphase II 2 cells, each going through meiosis II n each Each chromatid pair lines up at metaphase plate Cell 1 x2 Cell 2 Anaphase II 2 cells, each going through meiosis II n Cell 2 Cell 1 Cell 1a Telophase II The centromeres divide. Chromatid pairs separate and move to opposite poles of spindle Cell 2a 4 cells n The spindle disappears. Nuclear membrane and nucleolus reappear, and the cell splits. x2 At end of Telophase II Cell 2b Cell 1b Four sperm cells OR one egg cell and three polar bodies at n chromosomes each 4 cells n Figure 3 - Meiosis (continued): PROCEDURE 1. Examine a prepared slide of an ovary under the microscope. Low power will provide a broader field of view. 2. Identify the numerous primary follicles. These will appear as small round structures. Each contains a primary oocyte, egg cell (Greek `ōon = egg). 3. Find the larger secondary follicles. These structures contain the secondary oocyte, or egg, that has undergone meiosis I. When ovulation occurs, the egg is released from the ovary for movement down the fallopian tube. If sperm are present in the fallopian tube, fertilization and meiosis II may result. www.LabPaq.com 217 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis 4. Examine a prepared slide of a testis under the microscope. Low power will provide a broader range of view. 5. Identify the numerous seminiferous tubules that contain meiotically dividing cells. 6. Go to high power, locate one tubule, and observe the mature tailed sperm present in the tubule. www.LabPaq.com 218 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Exercise 3: Simulating Meiosis PROCEDURE Refer to Figure 3 while doing the following exercise. We will be simulating meiosis using pop beads to reinforce the sequence of events. 1. Interphase: a. Assemble the chromosomes: Count out eight each of the red and yellow pop beads (homologous chromosome pair colors, each representing a chromatid). The pop beads represent genes on the chromosome; each is composed of a three-nucleotide sequence, for instance, A-T-T, A-G-C, etc. b. Locate four plastic centromeres, the small tubes containing magnets. c. Connect a centromere in the middle of four red pop beads. Make two of these identical “chromatids” with two red beads on each side of each centromere. Each string of beads, connected at the centromere, is a chromatid. d. Connect a centromere in the middle of four yellow pop beads. Make two of these identical “chromatids” with two yellow beads on each side of each centromere. This will result in two yellow pop bead chromatids, connected at their centromere, to form the chromosome. e. Interphase for the single chromosome pair in the cell nucleus is complete. The beginning chromosome pair was a red and yellow strand, connected by their centromeres. You have just duplicated the chromosome pair, in anticipation of coming meiosis. 2. Prophase I: a. Form a “tetrad”, or foursome. The original chromosome pair is connected to the duplicated chromatids. All are connected at their centromeres. b. The nuclear membrane of the cell disintegrates, and the centrioles on the outside of the nuclear membrane move to opposite sides (poles) of the meiotic cell. Simulate the centrioles by placing two 1-inch pieces of a straw about two feet apart on a flat surface. Anchor them in place with tape. c. Spindle fibers begin to form. Cut two pieces of thread about 2 feet long. Run one piece of thread through each of the centrioles (straw pieces). Tie one end of the thread from one centriole to the red chromosome centromeres; then tie a thread end from the other centriole to the yellow chromosome centromeres. Temporarily separate the tetrad to tie in the spindle fibers, but then reattach the tetrad arrangement. www.LabPaq.com 219 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis 3. Anaphase I: a. Grasp the spindle fibers on the outside of the opposite centriole, and pull the chromosomes to the centrioles. The red chromosomes should pull to one centriole pole, and the yellow chromosomes to the other pole. b. NOTE that the original homologous red-yellow pair of chromosomes has now been split, with half of each pair being pulled to the opposite pole. This demonstrates the principle of independent segregation: For each homologous pair of chromosomes, the homologues separate from each other. 4. Telophase I: a. All spindle fibers disintegrate. Untie the centromeres. b. Nuclear membranes form around each separate set of chromosomes. The centrioles will replicate and be found as a pair just outside of the nuclear membrane. 5. Cytokinesis: Animal cells divide by forming a cleavage furrow and pinching off. Plant cells would divide by forming a cell plate, and splitting. Two cells result. Note: Meiosis is halfway completed. Each cell contains two chromosomes, either a red pair or a yellow pair, the original number. However, these two cells are genetically different from the original cell because they contain different homologues, or alleles, at each gene locus. Recall that the original pair was a red and yellow matched strand. 6. Prophase II, in each cell resulting from Cytokinesis above: a. Nuclear membrane disintegrates, and centrioles move to the opposite sides of the cell. Continue this exercise with one of the two cells, remembering that there is a second cell undergoing exactly the same steps. Use the green and blue beads to simulate the second cell’s activities. b. Tape down the centrioles in an area that represents the cell, with the centrioles located about two feet apart. c. Spindle fibers begin to form. Thread “spindle fibers” through the centriole, one per centriole. d. Place the two chromosomes, attached at their centromere, in the center of the cell, between the two centrioles. 7. Metaphase II: a. Tie one thread from one centriole to the centromere of one of the chromosomes. Tie the other thread from the other centriole to the centromere of the other chromosome. b. Reattach the two chromosomes at the centromeres. 8. Anaphase II: Hold the ends of the spindle fibers on the outside of the centrioles, and pull the respective chromosome to the centriole with the spindle fiber. www.LabPaq.com 220 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis 9. Telophase II: Untie the spindle fibers, as they are now disintegrating. 10.Cytokinesis: Animal cells now divide by forming a cleavage furrow and pinching off. Plant cells would divide by forming a cell plate, and splitting. Meiosis II is now complete. There are a total of four cells. Each cell contains one chromosome or n number of chromosomes. This has occurred for the formation of both the egg and the sperm separately. 11.Simulate fertilization by joining two n cells. Note that a 2n cell results with a pair of chromosomes, one from each n cell. That is what we began with at Interphase for both mitosis and meiosis. At this point, mitosis will increase the number of cells and a zygote will be formed, followed by the embryo stage. 12.Simulate Crossing-over (chiasma), a method by which recombination of genetic material occurs in chromosomes during meiosis. This commonly happens during Prophase I when segments of a chromatid may exchange places with identical segments on another non-sister chromatid. We can simulate this by exchanging identical sections of one of our red pop bead chromatids with an identical section of another yellow pop bead chromatid. Why would gene recombination not occur if we exchanged a red chromosome section with an identical red chromosome section? Now that you have completed this lab make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab write out a hypothesis for each exercise. www.LabPaq.com 221 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis Mitosis and Meiosis Margaret Vorndam, M.S. Version 42-0094-00-01 Lab Report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab reports by providing this information in an editable file which can be sent to an instructor. Observations Exercise 1: Mitosis in Animal and Plant Cells PROCEDURE 1. Observe the prepared slide of the whitefish blastula under the microscope. 2. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase, and Telophase. Place your drawings here: www.LabPaq.com 222 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis www.LabPaq.com 223 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis 3. For each drawing, label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cleavage furrow formation during Cytokinesis, and centrioles. It is unlikely that you will find all of these structures. Why? 4. Observe the slide of the onion root tip under the microscope. 5. Draw cells that represent the various stages of mitosis: Interphase, Prophase, Metaphase, Anaphase and Telophase. Place your drawings here: 6. Label the parts of the cells that you can identify such as the nuclear membrane, chromosomes, cellular membrane, cytoplasm, spindle fibers, chromatids, cell wall formation region, and centrioles. It it is unlikely that you will find all of these structures. Why? Results For both the whitefish and the onion, what is the 2n number of chromosomes that were observed? Hint: separate chromosomes are easiest to view and count during Prometaphase. questions A. What is the purpose of mitosis? B. What other term is commonly used in place of “mitosis”? www.LabPaq.com 224 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis C. What is a blastula? D. What are the differences in the mitotic processes between animal and plant cells? E. Why might there be more mitotic division in an onion root tip than in other areas of the plant? F. A summary of mitosis – fill in the correct answer. The nucleus in the undivided cell has the diploid (2n) number of chromosomes, and the nuclei in the two split cells also have _______ number of chromosomes. In mitosis, the chromosome number (check the correct choice) ___ stays the same or __ halves. Exercise 2: Meiosis in Animals questions A. Which organ, the ovary or the testes, contained the greater number of gametes? B. What may be a reason for this? Exercise 3: Simulating Meiosis Questions A. What does mitosis accomplish? B. When and where does mitosis occur? www.LabPaq.com 225 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis C. What does meiosis accomplish? D. When and where does meiosis occur? E. How do these two processes differ? www.LabPaq.com 226 ©Hands-On Labs, Inc. Experiment Mitosis and Meiosis F. How do Prophase in Mitosis and Prophase I in Meiosis differ? G. Does Meiosis occur anywhere else in the body beside within the gonads? H. How does meiosis confer advantages to an organism when compared to asexual reproduction such as budding? I. How might crossing-over be a benefit to an organism? J. How might crossing-over be detrimental to an organism? K. Crossing over can also occur during mitosis where chromosomes exchange segments. Is crossing over more advantageous to organism survival if it occurs during mitosis or meiosis? Explain. Laboratory Summary What have you learned from doing this laboratory? www.LabPaq.com 227 ©Hands-On Labs, Inc.
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