BIOL 102: Lab 8 Mitosis: Replication of Eukaryotic Cells PRE-LAB ASSIGNMENT: Students are expected to read pages 1-4 AND complete page 5 before coming to the lab to perform the experiments. Print this entire lab packet and bring it to the laboratory. Please provide a FULL lab report for this experiment following the “Lab Report Guidelines”. Objectives: By the end of this exercise you should be able to:  Describe events associated with the cell cycle.  Describe events associated with mitosis.  Distinguish between the stages of mitosis on prepared slides of mitotic cells.  Stain and examine chromosomes in mitotic cells.  Estimate the duration of the various stages of mitosis. Introduction: Have you ever watched a caterpillar turn into a butterfly? If so, you’re probably familiar with the idea of a life cycle. Butterflies go through some fairly spectacular life cycle transitions—turning from something that looks like a lowly worm into a glorious creature that floats on the breeze. Other organisms, from humans to plants to bacteria, also have a life cycle: a series of developmental steps that an individual goes through from the time it is born until the time it reproduces. The cell cycle can be thought of as the life cycle of a cell. In other words, it is the series of growth and development steps a cell undergoes between its “birth”—formation by the division of a mother cell—and reproduction—division to make two new daughter cells. Cell cycle To divide, a cell must complete several important tasks: it must grow, copy its genetic material (DNA), and physically split into two daughter cells. Cells perform these tasks in an organized, predictable series of steps that make up the cell cycle. The cell cycle is a cycle, rather than a linear pathway, because at the end of each go-round, the two daughter cells can start the exact same process over again from the beginning. In eukaryotic cells, or cells with a nucleus, the stages of the cell cycle are divided into two major phases: interphase and the mitotic (M) phase.  During interphase, the cell grows and makes a copy of its DNA.  During the mitotic (M) phase, the cell separates its DNA into two sets and divides its cytoplasm, forming two new cells. 1 Interphase Let’s enter the cell cycle just as a cell forms, by division of its mother cell. What must this newborn cell do next if it wants to go on and divide itself? Preparation for division happens in three steps:  G1 phase. During G1 phase, also called the first gap phase, the cell grows physically larger, copies organelles, and makes the molecular building blocks it will need in later steps. The cell continues to carry out its normal function.  S phase. In S phase, the cell synthesizes a complete copy of the DNA in its nucleus. Each chromosome consists of sister chromatids attached at the centromere (image to the right).  G2 phase. During the second gap phase, or G2 phase, the cell grows more, makes proteins and organelles, and begins to reorganize its contents in preparation for mitosis. G2 phase ends when mitosis begins. The G1, S, and G2 phases together are known as interphase. The prefix inter- means between, reflecting that interphase takes place between one mitotic (M) phase and the next. Mitosis Mitosis is a type of cell division in which one cell (the mother) divides to produce two new cells (the daughters) that are genetically identical to itself. In the context of the cell cycle, mitosis is the part of the division process in which the DNA of the cell's nucleus is split into two equal sets of chromosomes. The great majority of the cell divisions that happen in your body involve mitosis. During development and growth, mitosis populates an organism’s body with cells, and throughout an organism’s life, it replaces old, worn-out cells with new ones. For single-celled eukaryotes like yeast, mitotic divisions are actually a form of reproduction, adding new individuals to the population. In all of these cases, the “goal” of mitosis is to make sure that each daughter cell gets a perfect, full set of chromosomes. Cells with too few or too many chromosomes usually don’t function well: they may not survive, or they may even cause cancer. So, when cells undergo mitosis, they don’t just divide their DNA at random and toss it into piles for the two daughter cells. Instead, they split up their duplicated chromosomes in a carefully organized series of steps. Mitosis (M phase) usually last for less than 10% of the time of the cell cycle, which usually lasts 10 to 30 h. Actively dividing cells such as those in rapidly growing tissues may spend more than 10% of their time in mitosis, whereas static cells such as bone cells or neurons may rarely enter M phase. Binary Fission Prokaryotic cells lack nuclei and don’t undergo mitosis. Instead, they replicate their chromosome during a simpler process called binary fission (image below). 1. The DNA is replicated. 2. The cell elongates. 3. The DNA molecules move to opposite sides of the cell. 4. The membrane grows inward. 5. Two daughter cells are formed. 2 Cytokinesis Cytokinesis, the division of the cytoplasm to form two new cells, overlaps with the final stages of mitosis. It may start in either anaphase or telophase, depending on the cell, and finishes shortly after telophase. In animal cells, cytokinesis is contractile, pinching the cell in two like a coin purse with a drawstring. The “drawstring” is a band of filaments made of a protein called actin, and the pinch crease is known as the cleavage furrow. Plant cells can’t be divided like this because they have a cell wall and are too stiff. Instead, a structure called the cell plate forms down the middle of the cell, splitting it into two daughter cells separated by a new wall. In some tissues, cytokinesis is delayed or does not occur at all, and the cells are multinucleate. Some multinucleated tissues include striated muscle fibers and some algal filaments (see below). Nuclei Stages of Mitosis Mitosis (1) separates the genetic material duplicated during interphase into two identical sets of chromosomes and (2) reconstitutes a nucleus to house each set. As a result, mitosis produces two identical nuclei from one. Mitosis consists of four basic phases: prophase, metaphase, anaphase, and telophase. Some textbooks list five, breaking prophase into an early phase (called prophase) and a late phase (called prometaphase). The actual events of mitosis are not discrete but occur in a continuous sequence; separation of mitosis into four stages is merely convenient for our discussion and organization. These phases occur in strict sequential order, and cytokinesis - the process of dividing the cell contents to make two new cells - starts in anaphase or telophase. 3 Interphase Prophase Metaphase Anaphase Telophase Cytokinesis 4 LAB DATASHEET Activity 1: Simulate chromosomal replication and movement during mitosis Understanding the movements of chromosomes is crucial to understanding mitosis. You can simulate these movements easily with chromosome models made of pipe cleaners or popsicle sticks. This is a simple procedure, but a valuable one. 1. Examine the materials to be used as chromosome models provided by your instructor. 2. Identify the differences in chromosomes represented by various colors, lengths, or shapes of materials. Also identify materials representing centromeres. 3. Place a sheet of notebook paper on your lab table to use in representing the boundaries of the mitotic cell. 4. Assemble the chromosomes needed to represent nuclear material in a cell of a diploid organism with a total of six chromosomes. Place the chromosomes in the cell. 5. Arrange the chromosomes to depict the position and status of chromosomes during G1 phase of interphase. (During G1 the chromosomes are usually not condensed, as the chromosomes models imply, but the models are an adequate presentation). 6. Depict the status of chromosomes after completing S phase of interphase. Use additional “nuclear material” if needed. 7. Move the chromosome models appropriately to depict prophase. 8. Move the chromosome models appropriately to depict metaphase. 9. Move the chromosome models appropriately to depict anaphase. 10. Move the chromosome models appropriately to depict telophase 11. Draw the results of cytokinesis and the re-formation of nuclear membranes. 12. Chromosomal events occur as a continuous process of movements rather than in distinct steps. Therefore, repeat steps 4-11 as a continuous process and ask your instructor to verify your simulation. 13. Take a picture of the stage of your choosing and include it in your lab report. Be sure to label it. Mitosis in Animal Cells The most distinctive features of cellular replication in animal cells are the formation of asters with centrioles at their center (right) and cytokinesis. Cytokinesis includes formation of a cleavage furrow that begins on the periphery of the cell, pinches inward, and eventually divides the cytoplasm into two cells. Cells of a whitefish blastula provide good examples of the stages of mitosis and cytokinesis. Whitefish are commonly cultured fish whose eggs and early developmental stages undergo rapid cell division (as do all embryonic cells). A blastula is an early embryonic stage of a vertebrate and consists of a sphere of 25-100 cells with a high frequency of different mitotic stages. 5 Activity 2: Observe and describe mitosis in animal cells (Slide: White Fish Blastula) 1. Obtain a prepared slide of a cross-section through the blastula of a whitefish. 2. Examine the cells first on low (10x) then high (40x) magnification. Some of the cells contain condensed and stained chromosomes. 3. Refer to page 4 for a summary of the stages of mitosis. Identify examples of each on your prepared slide. Verify these stages with your lab partner or instructor/teaching assistant. 4. Also identify cells that you believe are between stages. 5. Examine the whitefish cells for signs of cytokinesis. 6. Prepared cross sections of cells show only two dimensions, but mitosis is a three-dimensional process. In the following space draw two cells in metaphase: one in which the cross section is parallel to the axis of the spindle apparatus and one in which the cross section is perpendicular to the spindle apparatus. Animal Cell Drawings of Mitosis: 6 Mitosis in Plant Cells Our model to study cellular replication in plants is the root tip of Allium (onion). Root tips of plants contain meristems, localized areas of rapid cell division due to active growth at the root tips. In plant cells, cytokinesis includes formation of a partition called a cell plate perpendicular to the axis of the spindle apparatus. The cell plate forms in the middle of the cell and grows out to the periphery. It will separate the two new daughter cells. Interestingly, the formation of the spindle apparatus and other microtubule systems in plant and fungal cells is organized by centrosomes, as in animal cells. But plant and fungal cells have no centrioles within the centrosomes. Thus, the function and necessity of centrioles remains somewhat of a mystery. Activity 3: Observe and diagram mitosis in plant cells (Slide: Onion Root Tip) 1. Examine a prepared slide of a longitudinal section through an onion root tip. 2. Search for examples of all stages of mitosis. Notice that most cells are in some part of interphase. 3. Search for signs of cell plate formation. 4. In the table below, diagram a plant cell with a diploid number of three pairs of chromosomes in each of the stages of mitosis. Diploid refers to a nucleus with two of each type of chromosome. Be sure to label the cell wall and cell plate. 5. Prepared cross sections of cells show only two dimensions, but mitosis is a three-dimensional process. In the following space draw two cells in metaphase: one in which the plane of section is parallel to the axis of the spindle apparatus and one in which the cross section is perpendicular to the spindle apparatus. Table 1: A diagram of a plant cell with a diploid number of three pairs of chromosomes in each stage Prophase Metaphase Anaphase Telophase 7 Plant Cell Drawings of Mitosis: 8 BIOL102: Introductory Biology Guidelines for Lab report preparation Fall 2017 • On the first day of lab, groups of 4 (maximum) will be self-assembled and fixed for the semester. • Lab reports must be submitted by the group (one report per group). o Every person in the group gets the same grade. o DO NOT include the names of individuals who did not attend the lab or help write the report. ▪ If you include the name of an absent individual, everyone will receive a zero. • Make sure to spell check and to use appropriate language and terminology. o Handwritten reports are not acceptable, unless otherwise stated. • Lab reports must be submitted using Blackboard in the corresponding folder (found in the Laboratory materials folder located in the Course materials section). • The report is via Blackboard a week after the experiments were completed. o Late reports will not be accepted. • There are a total of nine lab reports; the lowest non-zero grade will be dropped. The lab report must follow the following format: • Cover page: (Failure to include a cover page will result in a point deduction.) o Title o Date o Name of each active, present participant and the name of their professor next to it • Introduction (20 pts): o What is the topic of the lab? What is the theoretical background for the topic addressed in this lab? What are the goals? ▪ You may use information from the introduction. If you do, then paraphrase the information. DO NOT simply copy and paste from the lab sheet. Cite it in the references. • Materials and Methods (20 pts): o What are the materials/reagents/ instruments used in this lab? o What is the procedure followed? ▪ Explain the methods that you used to complete the lab which may vary from what is written. DO NOT simply copy and paste the information from the lab sheet. • Results (25 pts): o What observations did you make? Describe in words what you saw. ▪ Be precise, draw a diagram or insert a picture if it helps illustrate the point. • If you include either, then provide a figure legend or label it appropriately. o What measurements did you take (if applicable)? You can display these in a table. ▪ A picture of a datasheet is NOT acceptable if it is handwritten. ▪ Be sure to include ALL data and answers to all questions. • Conclusions (25 pts): o What did the results show? Explain what happened and why. o If the experiment did not work, explain what you think went work. • Literature cited (10 pts): o Provide the references of any sources that you used in the preparation of this report (for instance in the Introduction). You can also cite the pre-lab reading. o Wikipedia.org is unacceptable. General recommendations • Font and Organization o 12 pt Times New Roman Font o Double-spaced o Headings in bold font • Spelling and Grammar o Perform a spelling and grammar check because points will be deducted for excessive mistakes
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