Biology 214 ? Genes and Evolution ? Spring 2008 Lab #2: Mitosis and Meiosis Bring your text to lab. You will need to use it throughout the exercise. Review the following pages in Campbell et al. (2008): p. 231-233; 236: Phases of the cell cycle and mitosis p. 253-259: Meiosis PART I : MITOSIS Introduction Life continues on earth as a result of duplicating mechanisms at all levels, from the molecular stage to the whole individual, whether it be amoeba or man. When somatic cells (i.e., those cells which are not gametes) divide, they do so by a process known as mitosis. Growth and repair of tissue depends on this continued division of cells, which is the division of the nucleus of the cell and the precise distribution of the duplicated chromosomes between the two new cells. The cell cycle and chromosomes The division of nuclei by mitosis is exhibited by the somatic (body) cells in most plants and animals. During its life span, a cell passes through a regular sequence of physiological events called the cell cycle. This sequence includes several distinct stages, each characterized by certain metabolic activities of the cell. In actively dividing cells, the cycle may last only a few hours; in other cells, the cycle may last for days or weeks. At the completion of the cell cycle a new generation of cells is produced. The cycle is divided into four phases: G1, S, G2, and mitosis. The G1 (gap) phase is mainly a period when cytoplasmic materials are produced. New mitochondria, Golgi, ribosomes, and endoplasmic reticulum are formed. This is a period of active protein synthesis. The cells also grow in size during this period. Basically, this is the period when cells perform their normal function. The S (synthesis) phase is the time during which DNA synthesis occurs and the chromosomes are duplicated. During G2 phase, those structures directly related to mitosis (like spindle fibers) are produced. Collectively, G1, S, and G2 are referred to as interphase. Mitosis follows the G2 phase and involves the division of chromosomes and the formation of two new nuclei. Mitosis consists of four distinct stages, but actually takes up only about 5-10% of the complete cell cycle. The duplication of the genetic material (DNA) occurs in the nondividing cell prior to the initiation of mitosis. The chromosomes are already doubled when they become visible during prophase, the first stage of mitosis. The term mitosis refers specifically to the process of nuclear divi- sion, the orderly distribution of the chromosomes; between two daughter nuclei, starting with prophase and continuing through metaphase, anaphase, and telophase. Technically, therefore, mitosis occurs after duplication has been completed. Nuclear division usually occurs in close association with division of the cytoplasm (cytokinesis), but these two processes do not always occur together. Evidence from numerous experiments that have demonstrated that various chemical and physical treatments of dividing cells have different effects on mitosis and cytokinesis, clearly shows that different chemical and physical processes are involved in nuclear division (mitosis) and in cytoplasmic division (cytokinesis). An important point to remember is that cell division is a dynamic series of events during which the cell undergoes dramatic and often rapid physiological and morphological changes. The so- called stages of mitosis merely represent a few morphologically identifiable points in this continuum. 1 Preparing Live Onion Root Tips ? CAUTION: Wash your hands before and after lab. You may wear gloves while working with fixative (acetic acid and HCl). Do NOT pour fixative down drain?place in collection bottle for proper disposal. ? Aceto-orcein stain is permanent, so do not spill it. It can also be an irritant, so be careful not to breathe it in, and wash skin with soap and water in case of contact. ? Broken glass, microscope slides, and coverslips go in the cardboard sharps container. ? Paper items and gloves go in the regular garbage. 1. Working in groups of four, retrieve an onion that has been soaking and cut off one entire root with a razor blade. 2. Place the root in a 13 x 100 mm test tube, and pour in enough fixative (9 parts 45% acetic acid : 1 part Hcl) to cover it. 3. Incubate the tube for 6 min in a 50°C water bath. 4. Take the root out of the fixative with forceps and place it in the middle of a microscope slide. (Petri dishes will be available if you need to dump out the acid to retrieve your root tip.) Cut all excess from the root, leaving 2 mm at the growing end. (Make sure you leave the correct end, the one containing the apical meristem! Refer to pg. 747 in your text.) 5. Place 1 drop of aceto-orcein stain on the root tip and let soak for 2 min. 6. Use a kimwipe to carefully remove excess stain. Place a coverslip over the root tip. 7. Squash the root tip by applying slow, steady pressure to the coverslip with the eraser end of a pencil (or other implement). Squash the root tip straight down so as not to rupture or overlap the cells. Take care to not break the coverslip. 8. Place 1 more drop of stain along one edge of the coverslip. Draw the stain through the root tip by pressing a kimwipe to the other edge of the coverslip until the squashed tip is bathed in stain. Wait another 2 min. 9. Observe the cells under the microscope. Start with the lowest objective, then move up to the 40× ocular when you have identified an interesting area of the slide. 10. Compare your root tip preparation with the professionally produced root tip slide. If chromosomes are clearly visible in a large number of cells in your prepared slide, use your sample to complete the following exercises. If not, use the professionally produced slide. Observing Mitosis in an Onion Root Tip?Exercises 1. Using either your own onion root tip or a prepared slide, briefly sketch an onion root tip cell during each of the stages of mitosis as you see it on the slide. Use pages 232?233 and 236 of your text for guidance. Write a sentence or two explaining how you can tell that the cell is in this phase. Interphase:__________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ 2 Prophase:___________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ Metaphase:__________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ Anaphase:__________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ Telophase:__________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ ___________________________________________________ 2. Count the number of cells in your field of view that are in each phase of mitosis. Complete Table 1 to find an approximation of how many hours per day onion root tip cells spend in each mitotic phase. Table 1. Time spent in different phases of mitosis. Interphase Prophase Metaphase Anaphase Telophase Total Number of cells % cells (Number in each phase ÷ total) 100% Approximate time spent in each phase (% x 24 hrs) 24 hours 3 Questions: 1. Why do we use an onion root tip to observe mitosis? Why not any other kind of tissue? (See pg. 747 in Campbell et al., 2008.) ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 2. What can you conclude about the time spent in each phase of mitosis? Why do you think this is so? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 3. Would these results be different if we used a tissue other than the onion root tip? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Study the data in Table 2 and answer questions 4?5: Table 2. Minutes Spent in Cell Cycle Phases. Cell Type G1 S G2 Mitosis Beta 18 24 12 16 Delta 100 0 0 0 Gamma 18 48 14 20 4. Of the following, the best conclusion concerning the difference between the S phases for beta and gamma is that a. gamma contains more DNA than beta b. beta and gamma contain the same amount of DNA c. beta contains more RNA than gamma d. gamma contains 48 times more DNA and RNA than beta e. beta is a plant cell and gamma is an animal cell 5. The best conclusion concerning delta is that the cells a. contain no DNA b. contain no RNA c. contain only one chromosome that is very short d. are nerve cells e. divide in the G 1 phase 4 Take this moment to start your DNA isolation. Continue with Part II of the mitosis and meiosis lab during drying and incubation steps. Apply what you have learned about pipetting and centrifuging, but ask if you have questions. We will use the product later in the lab course. A. Isolation of DNA from cheek cells: NOTE: Anything that is used to isolate or touch human cells or DNA (including tips, eppendorf tubes and swabs/swab handles) must be disposed of as biohazardous waste into the red plastic containers labeled as such. You are not required to wear gloves, as you will be handling your own cells/DNA, but they will be made available to those who wish to wear them. 1. If you have eaten recently, first rinse out your mouth with water. Rotate a sterile swab inside your cheek for at least 30 seconds. 2. Let the swab air dry for approximately 15 minutes inside a clean 1.5 mL eppendorf tube. Be certain not to let the swab touch the bench or any other material. 3. Sign up for a sample identification number. Write this down! Label your tubes in the following steps with this number. 4. Pull off cotton tip of swab with a sterilized pair of forceps. To sterilize the forceps, dip the forcep tips into 95% ethanol, tap off excess ethanol and pass the tips through a bunson burner flame. [Do not leave forceps in the flame] Allow the flame to go out and the forceps to cool for a few seconds. Starting at the base of the swab, pinch the wooden handle firmly and twist the forceps around the handle while gently pushing up against the swab to release it from the handle. The more intact the swab is, the better your DNA yield. Place the swab tip into a 1.5 mL eppendorf tube. 5. Add 40 ?L of 0.2N NaOH (using a P200) 6. Heat in a 75°C waterbath for 10 minutes. 7. Add 360 ?L of 0.04 M Tris, pH 7.5 (using a P1000) 8. Mix by gently flicking the tube for several seconds. 9. Spin down the swab tip briefly in a microcentrifuge (be sure the rotor is balanced) 10. Pipet off 100 uL of the supernatant into a clean tube and label with your number, as assigned by your instructor. This is your isolated DNA. 11. Pipet off 100 ?L of the supernatant into a clean tube and label with your number, as assigned by your instructor. This is your isolated DNA. Discard the tube with the cotton swab in the biohazard container. B. Setup of PCR Reaction: 1. Use a micropipette with a fresh tip to add 25 ?L of your DNA to a clean 0.5 mL eppendorf tube (from the stock of 100?L you just prepared). 2. Use a fresh tip and add 20 ?L of the PCR MasterMix to your DNA. Mix by pipetting up and down 2 ? 3 times. 3. Label the cap of your tube with your number. In this way, your results will be anonymous. 4. Pulse your sample in the microcentrifuge (place the 0.5 mL tube inside a capless 1.5 mL tube so that your small tube does not get lodged in the centrifuge). 5. Store your sample on ice until your instructor is ready to begin the PCR amplification. 6. Your remaining isolated DNA (75 ?L) is often stored as a backup ? your instructor will have a labeled microfuge rack for your lab section ? make sure your number is written on the top of the tube. 5 PART II : MEIOSIS Introduction All cells of higher organisms do not divide in exactly the same manner as has just been described. Some, the germ or sex cells, undergo a special type of division which is known as meiosis. Meiosis is essential in the formation of germ cells; the general term for this process is gametogenesis; when it refers specifically to the formation of an egg, it is called oogenesis; and when it refers specifically to the formation of a sperm, it is called spermatogenesis. Meiosis is a specialized type of cell division that usually occurs during the formation of the gametes or sex cells of multicellular animals. During meiosis, the normal diploid (2N) chromosome number of the somatic cells is reduced by half to the typical haploid (1N) chromosome number of the gametes. Meiosis is extremely important for the survival and evolution of organisms because it provides for recombinations of genes during each generation upon which natural selection can operate to select the better adapted individual. Sexually reproducing organisms usually form male and female gametes at some point in their life history. Fertilization normally occurs at a later time in the life cycle and involves the fusion of male and female gamete nuclei. Thus, in order to maintain a constant number of chromosomes in successive generations (and to avoid doubling the chromosome number each time), some mechanism is necessary to provide a reduction (halving) of chromosome number between successive fertilizations. The process that results in the reduction in chromosome number is called meiosis. Meiosis generally consists of two successive nuclear divisions called the first and second meiotic divisions. Meiosis differs in two important respects from ordinary mitosis: ? The final number of chromosomes in a gamete resulting from meiosis is only half that of the parent cell, and each gamete or spore receives only one chromatid from each homologous pair of chromosomes that was present in the original parent cell. ? During the reduction in number, the chromosomes are assorted at random so that each gamete or spore receives a chromatid from one or the other member of each homologous pair. This random assortment of genetic material during meiosis plays a very critical role in the passage of traits from generation to generation. Homologous chromosomes are the paired chromosomes found in diploid cells that are very similar in size and shape but differ both in origin (one comes from the father and the other comes from the mother) and in genetic composition, as the mother and father usually do not have the exact same set of alleles. Meiosis, like mitosis, is a dynamic process during which the cells are undergoing continuous changes. Nonetheless, a good understanding of the process can be achieved by describing it as a sequence of two nuclear divisions, each with four distinct stages and with an intervening interkinesis stage between the first and second meiotic divisions. Meiosis simulation with pop bead models 1. Before you begin, it will help to review the following terms and concepts: ? diploid ? haploid ? homologous chromosomes ? chromatid and sister chromatids ? tetrad ? In what type of cells does meiosis occur? Why? 2. Begin the meiosis simulation with a parent cell containing 2N = 4 chromosomes (2 homologous pairs, one large pair and one small pair). Each lab group should construct the chromosomes as follows: 6 a) For the large chromosome, attach a string of five beads of one color to one side of a centromere and repeat for the other side (Fig. 1). b) Repeat step a with beads of the other color to make the homolog. c) Repeat steps a and b for a pair of small chromosomes using a total of 5 beads per chromosome. Figure 1. Two homologous pairs of chromosomes at the start of the meiosis simulation. 3. Using your text as a guide, model the chromosomes during the steps of the cell cycle in interphase, meiosis I, and meiosis II. A piece of paper can represent the cell. Each member of the group should demonstrate the stages of meiosis while describing them using the correct terminology to the other group members. 4. When everyone can do this, show your instructor or UTA and proceed to the application questions. Questions: 1. Take a moment to observe the gametes resulting from other simulations. What are the two processes that contribute to genetic variation during meiosis? When do each of these occur? (See pg. 258 & 259 in Campbell et al., 2008 if you are struggling) _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ Refer to figure 2 to answer questions 2-5. You may want to start by labeling the phases of the cell cycle and meiosis on the diagram. Figure 2. Diagram of a meiotic process. Time 1x 2x 3x 4x DNA c o n t ent /c el l I II III IV VDNA c o n t ent /c el l DNA c o n t ent /c el l 7 2. Which number represents G 2 ? a. I b. II c. III d. IV e. V 3. Which number represents the DNA content of a sperm cell? a. I b. II c. III d. IV e. V 4. Which number represents the separation of homologous chromosomes? a. I b. II c. III d. IV e. V 5. Where would you place crossing over in this diagram? a. I b. II c. III d. IV e. V Use Figure 3 to answer questions 6 and 7. Figure 3. Diploid cell with four chromosomes. There are two types of chromosomes, one long and the other short. The dotted lined chromosomes came from the egg and the solid lined chromosomes came from the sperm. At this time, the chromosomes have not yet replicated. 6. A possible end result of the cell in Figure 3 undergoing Meiosis I is: C. A B D. Centromere 8 7. A possible end result of Meiosis II is: B D.A C. ___________________________________________________________________________ For next session: ? Print out and read Lab 3 protocol ? Bring in potential mutagens and a primary research article for Ames Lab ? Read Pechenik pgs 160-166 (writing a methods section); 206-216 (writing an introduction) ? Prepare for quiz 1, covering: o Lab 1 macromolecules o Lab 2 mitosis and meiosis o Lab 3 demonstrate that you understand the Ames lab protocol o Pechenik readings assigned for labs 1-3 o lab techniques 9 Sarah Rose Carrino Week 5
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