LABORATORY. o Name, identify, and describe the events occurring during the four phases of the cell cycle.

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1 LABORATORY OVERVIEW According to the cell theory, all cells come from preexisting cells. New cells are formed by the process of c~1i division, which involves both division of the cell's nucleus (karyokinesis) and division, of the cytoplasm (cytokinesis). There are two types of nuclear division: mitosis and meiosis. l'1itosis typically results in the production of two daughter nuclei that are genetically identical to each other and to the parent nucleus. Formation of an adult organism from a fertilized egg, asexual reproduction. regeneration, and maintenance or repair of body parts are all accomplished by mitotic cell division. Meiosis, on the other hand, reduces the chromosome number in daughter nuclei to half that of the parent cell. Gametes (sex cells) in animals and spores in plants are both produced by meiotic division. During this laboratory period you will focus on mitosis; you will investigate meiosis in Laboratory 13. STUDENT PREPARATION Prepare for this laboratory by reviewing the text pages indicated by your instructor. Do Exercise A before coming to the laboratory and familiarize yourself with the contents of Exercises B through D. I1III Objectives o Name, identify, and describe the events occurring during the four phases of the cell cycle Procedure The term cell cycle is used to describe the life history of actively dividing cells. Repeated cellular divisions are separated by periods of growth and preparation for the next division. Even though it is a continuous process, the cell cycle can be separated into several stages (Figure 9A-l). After a cell divides, it enters interphase, which consists of three stages: G I, 5, and G 2 Interphase is often referred to as a "resting" stage, but cells in interphase are not really at rest. Indeed, they are actively preparing for the next division.

2 9-2 Laboratory 9 Figure 9A-1 Stages of the cell cycle. G 1 and Gz stand for the first and second "gaps" in the cell cycle. S stands for synthesis of DNA. Mitosis is also called" M phase." G} phase G2phase Figure 9A-2 Electron micrograph of an isolated chief cell from mouse gastric mucosa. Note the darkly stained chromatin around the inner surfaceof the nuclear membrane. During interphase, DNA, with its chromosomal proteins, exists in a highly uncoiled state. Thus, when cell contents are stained, distinct chromosomal structures are not visible at this stage. Chromosomes appear, instead, as a granular material called chromatin within the nucleus (Figure 9A-2). '0 0, Events of G I During G 1 (first "gap"), the cell approximately doubles in size, and its enzymes and organelles double in number. a. Why are the activities of G 1 important for a cell that is preparing to divide? j Late in the G 1 period, a critical restriction point is reached that determines whether or not the cell will continue to divide. If the cell does not proceed to the S phase, it exits the cell cycle and becomes "non-dividing." Nondividing cells are considered to be in the Go phase. Most cells of the body share this fate. Some, such as nerve cells, may never divide again; others can resume dividing if necessary. An example of the latter is the remarkable ability of the liver to regenerate after damage or transplant. In order to pass the restriction point, a cell must reach a certain size. The ratio volume of cytoplasm/ genome size appears to be of most importance. Other environmental factors such as cell density or adherence to a substrate may also playa part in regulation at this point in the cell cycle.

3 Mitosis 9-3 In addition, protein kinase enzymes fluctuate in concentration throughout the cell cycle. Kinase enzymes control cell cyele activity by phosphorylating specific proteins. Regulatory proteins, called cyelins, can in turn regulate protein kinases. Protein kinases controlled by cyelins are called Cdk proteins (cyelin-dependent kinases). A specific cyclin-cdk enzyme complex is required for cells to pass the restriction point. Events of 5 Before the S phase, each chromosome consists of a double-stranded helix of DNA. During the S phase, the two strands of the DNA helix unwind and separate, and each duplicates by a process called replication. By the end of the S phase, each chromosome is composed of two helices of DNA, called chromatids, joined at a region of the chromosome known as the centromere (Figure 9A-3). Figure 9A-3 Replication of chromosomal DNA. The two strands of a helix of DNA separate, and each sister chromatids directs the synthesis of its other of a chromosome DNA halfso that each chromatid will be chromosome replication chromatid chromatid composed of a double-stranded helical DNA molecule. The small oval shape ~ located at the centromere region is a chromosome centromere{ kinetochore---a small proteinaceous region body to which spindle fibers attach during mitosis. 2 S '\ r S Gz Mitosis daughter original DNA newly synthesized DNA daughter chromosome Distinct chromosomes are not yet visible during this phase. The DNA molecules are structurally intact, but are largely uncoiled and dispersed as chromatin. Chromosomal proteins, also synthesized during the S phase, will eventually associate with DNA to help coil it into tightly packed chromosome structures prior to the onset of mitosis. b. Why is it necessary for DNA to duplicate before cells divide? Events of G 2 During the G z phase, structures involved directly in cell division are synthesized. Spindle fibers begin to assemble. These will become attached to chromosomes to guide their movement during cell division. In animal cells, a pair of centrioles completes division to form two pairs of centrioles. These will also playa role in the movement of chromosomes during the mitotic process. Cells of higher plants have spindle fibers but usually lack centrioles. Regulatory proteins also serve an important role in the transition from the G z phase to the M phase (mitosis). This transition is regulated by a buildup of cyelins at the end of the Gz phase and their regulation of a Cdk protein kinase as part of an enzyme complex known as MPF (M-phase promoting factor). At the end of the M phase, MPF activates an additional enzyme that destroys cyelin and turns off MPF activity. c. Summarize the events of interphase in the following figure. «

4 9-4 Laboratory 9 ---Events G} phase -Events G2phase L--_~ Events Objectives ft i iii i i Ii III! II o Relate the process of DNA replication to the process of mitosis. o Describe the structure of a chromosome in late prophase and identify its parts. o Name, identify, and list the events occurring during the stages of mitosis. o Describe the functions of centrioles, centromeres. and spindle fibers in mitosis. o Compare the process of cytokinesis in animal and in plant cells Procedure H lib II nil illil The purpose of this exercise is to simulate major features of mitotic cell division, not to duplicate it. The materials used will limit how closely you can approximate the actual process. On your laboratory bench, you will find a chromosome simulation kit consisting of two strands of red beads and two strands of yellow beads, four hollow plastic cylinders, and four pieces of string. The strands of plastic beads represent chromosomes. The magnetic piece in each"chromosome" represents the centromere region. The four hollow plastic cylinders represent centrioles. The string will be used to simulate spindle fibers. ~ Figure 9B-l shows a simulation of a chromosome as it would appear before the S phase of interphase, when DNA duplication occurs. It represents one double-stranded helical DNA molecule. Figure 9B-2 shows a simulated chromosome as it would appear after DNA replication. Each half of this duplicated chromosome is called a chromatid and contains its own helical DNA molecule. The two chromatids attached at the centromere region are identical and are called sister chromatids. Figure 98-1 Early interphase. A chromosome, as it appears before DNA duplication in interphase, is composed of one double-stranded DNA molecule (see Figure 9A-3, G 1 phase).

5 a. Next to each diagram (Figures 9B-1 and 9B-2), draw its representative DNA structure. Is the helix doublestranded or single-stranded in each chromatid? Mitosis 9-5 Figure 98-2 Mid-interphase. A chromosome as it appears after DNA duplication interphase. Each sister chromatid contains onedouble-stranded DNA molecule (see Figure 9A-3, Gz phase). Interphase Place one strand of red beads and one strand of yellow beads near the center of your work area. Together these two strands of beads represent a homologous pair of chromosomes." b. What is the significance of using two different colors for the two homologous chromosomes? Could you have used two red strands, one short and the other long? Why or why not? Keep in mind that chromosomal DNA would not be seen in the form of chromosomes at this stage, but would exist as part of the diffuse chromatin contained within the nucleus. Position two of the hollow plastic cylinders "centrioles") at right angles to each other outside an imaginary nuclear membrane or envelope surrounding the chromosomes. DNA synthesis occurs during interphase to produce chromosomes consisting of two chromatids. Simulate this process by bringing the magnetic centromere of the second red strand on the table. Do the same with the yellow strands. Two chromosomes, each consisting of two sister chromatids, are now before you (Figure 9B-3). Simulate centriole replication by placing two additional plastic cylinders next to the original centriolar bodies. c. Why do you need to replicate the centrioles? Figure 98-3 Late interphase. CJ CJ Chromosomal DNA hasalready centrioles duplicated and the centrioles have o 0,.., replicated by the end of interphase. / " "" nuclear The dashed line in (a) represents the / \ envelope I \ nuclearenvelope. I \ I \ nucleolus I I I I f-f chromatin \ I \ I \ I cytoplasm \ / "- -.,.." / <,_--_... cell membrane (a) (b) 'In diploid organisms, chromosomes of somatic cells occur in matched pairs that resemble each other in size, shape, and location of their centromeres. Matched pairs are called homologous chromosomes. One member of each pair is paternal and the other, maternal, in origin.

6 9-6 Laboratory 9 Mitosis Prophase During prophase, chromatin condenses within the nucleus until the chromosomes and their individual chromatids are visible. The two pairs of centrioles, replicated during interphase, separate and migrate to opposite sides or poles of the nucleus. A dense material surrounding the centrioles is associated with microtubule formation and is often referred to as a microtubule organizing center. This material, in combination with the centriole pair at its center, is called a centrosome. Spindle fibers composed of microtubules begin to form. The assembly of microtubules is initiated by the centrosomes and requires the polymerization of globular tubulin protein molecules into fibers (similar to lengthening a string of beads). As the spindle fibers lengthen, the nuclear membrane and nucleoli disappear. The spindle fibers attach to a protein structure, the kinetochore, located at the centromere region of two sister chromatids. Kinetochore microtubules extend from the centrosomes to the kinetochores of chromosomes. Some fibers reach all the way across the cell from centrosome to centrosome, while others seem to stop short of attachment. Kinetochore microtubules shorten during anaphase by depolymerization of tubulin fibers at their kinetochore ends. Begin your simulation of the events of prophase by separating the two pairs of cylindrical beads, moving one pair to the opposite side of the imaginary nuclear membrane that surrounds the chromosome bundle. Approximately 50 em from each side of the chromosomes, tape down one centriole of each pair so that it is pointing toward the nucleus (Figure 9B-4). Figure 98-4 Early prophase. Centrioles have migrated to opposite sides of the nucleus and spindle fibers will soon become attached to the centromere regions of the chromosomes. Form a loop at one end of each piece of string in your kit. Draw the loop of one piece of string tightly around the centromere region of one of the sister chromatids (Figure 9B-5); repeat this process for the other sister chromatid and for the sister chromatids of the homologous chromosome. Extend the strings (spindle fibers) of sister chromatids toward opposite sides of the nucleus; thread each string through one of the cylinders (centrioles). Figure 98-5 Attachment of spindlefibers to the centromere regions of sister chromatids. T '.....::,...,.':

7 ~ Mitosis 9-7 Metaphase During metaphase, the centromere regions of sister chromatids are attached by spindle fibers to opposite centrioles and the chromosomes line up in single file at the middle, or equator, of the spindle apparatus (Figure 9B-6). d. Why is it important that the chromosomes line up in single file during metaphase? Figure 98-6 (a) The mitoticspindle apparatusof animal cells consists of astral fibers (the short fibers that surround the centrioles), spindle fibers that attach to chromosomes, and spindle. fibers that extend between centrioles. (b) In cells of higher plants, the mitotic spindle apparatus has spindlefibers but lacks centrioles and astral fibers. (a) I 1 f.:.'..1 To simulate metaphase, center the chromosomes between the centrosomes (Figure 9B-7). Figure 98-7 Metaphase. Chromosomes line up in single file on the mitotic spindle apparatus. Chromosomes appear to be perpendicular to the long axis of the spindleapparatus and areequidistant from each pole of the cell. (b) I.,

8 ~. k j' 9-8 Laboratory 9 Anaphase J: chromosomes? During anaphase, the sister chromatids of each chromosome separate at the region of their centror{ and move toward opposite poles. Recall that the spindle fibers shorten at their kinetochore ends and the chromosomes follow. Thus they appear to be pulled toward opposite sides of the cell The separated chromatids are now called daughter chromosomes. The name of each of the two DNA strands, formerly held together at the kinetochore, has changed from chromatid to chromosome. When a strand of DNA has its own centromere region, it is called a chromosome. To simulate anaphase, pull on the strings until the magnetic centromeres are separated. Continue pulling the daughter chromosomes toward the centrioles. Note that the arms of each chromosome follow the centromere toward the pole (Figure 9B-8). e. During mitosis, when would you change the description of DNA strands from chromatids to Figure 98-8 Anaphase. Centromere regions of sister chromatids separate, and daughter chromosomes move to opposite ends of the cell. >: 1 ":.l'; Telophase The spindle apparatus disappears during telophase and nuclei are re-formed as the nuclear membra:l1ef~, appears. Nucleoli also reappear. There are now two nuclei, one for each of the daughter cells...' Chromosomes decondense and re-form diffuse chromatin. f. How many helices of DNA are in each of the daughter chromosomes at this time? To simulate telophase, remove the strings. Near each pair of centrioles, pile up the one red and:~,:'; yellow chromosome drawn to that side of the cell during anaphase (Figure 9B-9). These representtlf daughter nuclei and daughter cells. Note that you began cell division with two chromosomes: a reg' and a yellow one. 1-;;;, '. -.,.';~~/~~. Figure 98-9 Telophase. Sister chromatids haveseparated, forming daughter chromosomes, and new nuclear membranes form aroundthe separated chromosomal bundles. g. How many chromosomes do you have in each of your daughter nuclei? Note: The number of chromosomes in a cell is determined by counting the number of visibly sepa~ centromeres, regardless of whether the centromere region joins two sister chromatids in a chroffiq, appears in an undivided chromosome composed of only onechromatid., Repeat the procedure outlined above until you are thoroughly familiar with the major events~, mitosis. Have your instructor check your work if you experience difficulty...

9 Mitosis 9-9 I After you are comfortable with this simulation of the mitotic process, combine your chromosome kit with that of another student. Shorten the two yellow and two red strands from one of the kits so that there are only five beads on each side of the centromere region. Now simulate mitosis in a nucleus with four chromosomes (two homologous pairs, one pair consisting of one short red and one short yellow chromosome, and the other of one long red and one long yellow chromosome). Draw each stage (prophase, metaphase, anaphase, and telophase) on a separate piece of paper and insert this into your laboratory manual. You should end up with two nuclei, each containing four chromosomes-two red and two yellow. Have your instructor check your work. When you are finished, reattach the beads to the original chromosomes and separate the two kits in preparation for the next class. Cytokinesis Division of the cytoplasm (cytokinesis) usually occurs as telophase of mitosis progresses. In animal cells, microtubules playa role in the formation of a furrow that constricts the cytoplasmic mass into two daughter cells (Figure 9B-10a). In plant cells, a cell plate is formed along the midplane of the dividing cell as many vesicles (produced by Golgi bodies) become joined together. This creates a membrane-bound space, the cell plate. Membranes of either end of the cell plate fuse with the cell membrane to produce two separate daughter cells. The area between the cells becomes impregnated with pectin to form the middle lamella, and the two daughter cells synthesize cell walls (Figure 9B-10b). h. How does the number of chromosomes in the two daughter cells that you formed in your simulations compare with the number of chromosomes in the parent cell? i. How do the two cells that you formed in your simulation compare with the parent cell with regard to which chromosomes are present? (a) cell plate (c) (b) Figure plant cell. (a) Cytokinesis in an animal cell. (b) and (c) Cytokinesis in a

10 I. D' n..-.'.'if.,ti!1!ltllllrlnrtunn ,. 8 ~.~ ~. _ Laboratory 9 ;. Would the constitution of the daughter cells be any different if you had lined up the metaphase chromosomes in a different order? ( k. If the red chromosomes represent paternal chromosomes and the yellow chromosomes represent maternal chromosomes, what can be said about the genetic constitution of the daughter cells? 1. Keeping your answer to the previous question in mind, briefly describe how mitosis maintains a constant chromosome number and why daughter nuclei arealways genetically identical. II ai! Objectives!! U !llIllllllilili II BgH lilli I a! o Identify the stages of mitosis in living tissues from the onion root tip. U Relate the apparent frequency of mitotic figures to the growth pattern of root-tip tissue. I BII B Procedure i I I11 I1I11 III II! III i IIII1I Mitosis is easily studied using the root tips of actively growing plants. Roots of the common garden onion (Allium cepa) provide good material for such a study (Figure 9C-1). (Allium cepa has 16 chromosomes.) 1. Obtain a stained root tip for microscopic examination and place it on a slide in a drop of water. Add a coverslip, gently pressing downward with the eraser end of a pencil to spread the tissues apart. 2. Observe the preparation using a microscope (lox) and squash it further if necessary; do nul be too heavy-handed initially. Figure 9C-1 Median longitudinal section of an onion root tip. region of elongation apical ~---'--'-----meristem root cap 3. Use the microscope to examine the Allium root-tip slide. Scan the entire length of the slide to observe cells far from the tip and cells right at the tip. Are the cells in these areas dividing? Locate the area of cell division, the region where the greatest number of mitotic figures are represented, and draw a box around this area in Figure 9C-1. Label this area "region of cell division."

11 r Mitosis I 9-1 1, At the apex of the region of cell division, certain cells called "initials" give rise to new cells for root growth. When an initial cell divides, one daughter cell goes on to divide a few i" more times (in the region of cell division) before differentiating into specialized cells. The 1 r other daughter cell continues as an initial cell and remains in the apical region (called the apical merisiem; meristem refers to undifferentiated plant tissue from which new cells are formed). Covering the apical meristem is the root cap. Behind the region of cell division, cells elongate and later differentiate as the root lengthens. a. What might be the purpose of the root cap? 4. Switch to high power (40X). Use Figure 9C2 to identify cells in interphase, prophase, metaphase! anaphase! and telophase. Draw and label these cells on a separate sheet of paper. Insert the drawings into your notebook. Figure 9C-2 Stages of mitosis in the onion root tip. interphase prophase metaphase anaphase telophase EXERCISE D" Now that you are familiar with the events of mitosis and the stages of the cell cycle, you can estimate the relative duration of each phase of the cell cycle by recording the frequency with which you find each phase in regions where cell division is actively taking place. The frequency of a phase is an indication of the relative length of that phase Objectives III III III II! III o Use observations of cells in an onion root tip to describe the relative duration of the phases of the cell cycle. I1111 Procedure 1111I I111 i II 1. Obtain a prepared slide of onion root tip or use your fresh onion root-tip squash if the preparation is good and many mitotic figures are visible.

12 9-12 Laboratory 9 2. With the 40X objective in place, examine a single field of view (the area visible in the microscope with the slide stationary) in the apical meristem region and count the number of cells in tr phases of the cell cycle listed in Table 9D-1. Make sure you are surveying the actively divl 6 area of the onion root tip. Repeat this count in at least two more nonoverlapping fields. Use Table 9D-1 to collect and calculate your results. Table 90-1 Percentage of Cells in Each Phase of the Cell Cycle Number of Cells Field 1 Field 2 Field 3 Total Percent of Grand Total (Total/Grand Total X 100) Interphase Prophase Anaphase Telophase Grand total ><><>< The duration of mitosis varies for different tissues in the onion. However, prophase is always the longest phase 0-2 hours), and anaphase is always the shortest (2-10 minutes). Metaphase (5-15 minutes) and telophase minutes) are also of relatively short duration. Interphase may range from 12 to 30 hours. Consider that it takes, on average, 16 hours (960 minutes) for onion root-tip cells to complete the cell cycle. You can calculate the amount of time spent in each phase of the cei, cycle from the percentage of cells in that stage: Percentage of cells in stage X 960 minutes = minutes of cell cycle spent in stage 06 hours) 3. Calculate the following (convert the times to hours and minutes): Time spent in: prophase hr min metaphase hr nun anaphase hr min telophase hr min Total time spent in mitosis hr min Time spent in interphase hr min a. What percentage of the cell cycle is spent in mitosis? In interphase? b. How do your results compare with what is generally known about the Allium cepa cell cycle, as described above? c. If your data show little agreement with the information given on the Allium cepa cell cycle, w,, the cell cycle for the root-tip cells you observed have to be longer or shorter than the average 0/ 16 hours in order to explain your results?

13 Mitosis If you are using a prepared slide of an onion root tip, you will be observing a longitudinal section through the root tip. d. How does a longitudinal section compare with a cross section? e. Why do some of the cells appear to be empty? _ 1. The diagram below shows the relative amount of DNA present in a cell progressing through the stages of the cell cycle. Label the line segments between the hatch marks to show GIl S, G 21 and mitosis. 2 I I.' 2. Complete the following table. time G] 5 Number of Chromatids per Chromosome G 2 Prophase Metaphase Anaphase Telophase 3. How do protein kinases and cyclins regulate the cell cycle?

14 9-14 Laboratory 9 4. What role does cell division play in the growth of an organism? 5. What is the function of the centromere region of a chromosome? 6. What is a centrosome? How are centrioles and centrosomes related? 7. What is the function of spindle fibers? How do they lengthen and shorten? 8. When does a chromatid become a chromosome? 9. You have a diploid cell containing eight chromosomes (2n = 8). What would this cell look like at metaphase? How many chromosomes would each of the daughter cells have? 10. You are given two dishes of beads, one containing red beads and the other yellow. Describe how you would simulate the chromosomes of a diploid cell for which 2n = Can a haploid cell undergo mitosis? Why or why not? If you had a haploid cell with four chromosomes in = 4), what would it look like during metaphase? 12. Indicate the stage of mitosis (interphase, prophase, metaphase, anaphase, telophase) during which each of the following events occurs. Nuclear membrane disappears. Centrioles replicate. Chromosomes are arranged in single file between the poles of the cell. Chromatids separate. Spindle fibers form. Cell plate forms. DNA replication occurs. Chromosomes first become visible as long thin strands. Chromosomes move to opposite poles of the cell. b-,8>.b :itit&. #,6..& d...