life Lab 7 Centromere region One (replicated) chromosome Sister Figure I. The Cell Cycle. Figure 2. A Replicated Chromosome.

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1 71 life.. -' - \ Lab 7 Cell Division Cellular reproduction in the cells is accomplished by mitosis or meiosis. The chromosomes of the cell have to repli cate themselves in both processes and then move away from each into new cells. Mitosis results in two daughter cells that are identical to each other as well as the original parent cell. Somatic, or body cells, undergo mitosis. Cells begin with two sets of chromosomes and end with two sets of chromosomes. Meiosis results in four daughter cells that are different from each other as well as different from the parent cell. The cells that are produced by meiosis arc called gametes (sperm or eggs) and have only one set of chromosomes. Both types of cell division fall into the same phase in the cell cycle (Figure 1). They are not processes that can occur simultaneously in the same cell. As soon as mito sis or meiosis has occurred then it is followed by cytokinesis, which is the division of the cytoplasm. One (replicated) chromosome Centromere region Sister chromatid Sister chromatid Figure I. The Cell Cycle. Figure 2. A Replicated Chromosome.

2 72 Terminology 1. Chromosome DNA (deoxyribonucleic acid) molecule with associated proteins (Figure 2) 2. Chromatid result of chromosome replication (two sister chromatids make up a chromosome) (Figure 2) 3. Centromere region where the sister chromatids are joined (Figure 2) 4. Ccntrosomes made of centriole pairs found at the polar regions of the cell; also called the MTOC (microtubule organizing center) because it is thought to organize the spindle fibers used for mitosis or meiosis; not present in most plants 5. Spindle fibers made of tubulin and used to move the chromosomes during the M phase 6. Kinetochore structures next to the outside of the centromere to which the spindle microtubules attach on each sister chromatid 7. Haploid cells that contain one set of chromosomes («) 8. Diploid cells that contain two sets of chromosomes (2/i) 9. Cleavage furrow formed by a contractile ring of actin that tightens until the cell pinches into two cells 10. Cell plate formed in plants instead of a cleavage furrow to help form the cell wall; this is formed from the inside to the out 11. Homologous in a diploid cell with two sets of chromosomes each chromosome will have its matching chro mosome to complete that set (2 of chromosome 1, 2 of chromosome 2, 2 of chromosome 3, etc.); one chro mosome of the set is maternal and the other is paternal 12. Tetrad a pair of homologous chromosomes Phases of mitosis Phases of meiosis 1. prophase 1. prophase I 6. prophase II 2. proinctaphase 2. prometaphase 1 7. prometaphase II 3. metaphase 3. metaphase I 8. mctaphase II 4. anaphase 4. anaphasc I 9. anaphasc II 5. telophase 5. tclophase I 10. telophase II Activity A. Mitosis Simulation Keep in mind that mitosis is a continuous process. Scientists have named obvious stages that make it easier to study this process. The end result of mitosis will be two identical cells that have the same chromosome number as each other and the parent cell from which they came. A diploid cell will produce two diploid daughters (Figure 4). Your hypothetical cell will be 2/;=4 or having two pairs of homologous chromosomes. Let the paternal chromosomes be blue and let the maternal chromosomes be red. Pick a different bead color for the centromeres and centrosomes. 1. Make the maternal chromosomes 10 beads and 4 beads long. The 10 bead chromosome will have 5 red beads on each side of the centromere and the 4 bead chromosome will have 2 red beads on each side of the cen tromere (Figure 3).

3 73 2. Repeat step 1 using blue instead for the paternal chromosomes. There should be a total of 4 chromosomes when you are finished Make a centriole pair by attaching 2 beads of a different color to each other. Using a very long piece of string, make a cell and place your bead struc tures inside. Assume that a nucleus is present, but we will not diagram one during this exercise. You are going to proceed through the stages of the cell cycle. S phase and G2 phase Replication of the chromosomes occurs in the S phase and the centrioles finish replication in the G2 phase. Make 4 more chromo somes just like your originals and also make another centriole pair. Prophase The centrosomes migrate to opposite ends of the cell as the spin dle apparatus starts to form between them. The chromosomes shorten and thicken and the nuclear envelope disassembles. Using additional pieces of string show the spindle. Prometaphase The spindle apparatus is completely formed with the cen Figure 3 Hypothetical Cell with Four Chromosomes Reproduced from A Ijitwratiiry Guide to thr b'aturul Wtrtd. (2003) by pcrmkiian uf Picnlicc-Hill. Inc. trosomes placed opposite of each other along the width of the cell. This phase is where the spindle fibers attach to the kinetochores of the chromosomes and start to move the chromosomes toward the metaphase plate (equatorial plate). Metaphase Each chromosome lines up along the metaphase plate with one kinetochore facing one pole while the other kinetochore faces the opposite pole. This creates a tension that allows for the signal to be given that indicates the start of anaphase. Anaphase Immediately after the chromosomes line up in metaphase the spindle fibers will pull the sister chromatids apart from each other toward their respective centrosome. They are now considered chromosomes again after the sister chromatids have been split apart. Telophase Once the chromosomes reach the centrosomes and stop moving then this phase will begin. Cytoki nesis begins at this time and the spindle begins to disintegrate. Towards the end of telophase the nuclear enve lope will reappear. Your resulting daughter cells should look identical to the cell that you started with in terms of chromosome and centriole number. Activity B. Meiosis Simulation Germ cells are the only cells that undergo meiosis. The end product of meiosis will be four gametes (sperm or eggs). Sex ual reproduction requires the union of the sperm with the egg to form a new cell that will undergo mitosis to form a new multicellular organism. Humans have 23 pairs of chromosomes («=23 or 2/?=46) that make up their genome. Keep in mind that one set is maternal and one set is paternal. The gametes will be haploid to prepare for fertilization, which restores the normal somatic diploid condition. If the gametes were not haploid, then the resulting cell from fertilization would be double the normal somatic chromosome number. In the case of humans, the total number of chromosomes would be 96 chromosomes. Meiosis resembles mitosis in some ways. Meiosis consists of two main stages of division, meiosis I and meiosis II. Replication of the chromosomes will occur only once before the start of meiosis I (Figure 5).

4 74 MltQSISANP CYTOKINESIS replicated, uncondensed DNA i chromosomes (each a pair of sister chromatids joined together) - pair of nucleus nucleolus centrosomes spindle fibers (microtubules) mitotic spindle metaphase plate END OF INTERPHASE DNA has already duplicated back in the S phase. Centrosome has doubled. PROPHASE Mitosis begins. The chromosomes take shape as the DNA condenses. The nuclear envelope begins to break down. The two pairs of centrosomes begin to move toward the cellular poles, sprouting microtubules as they go. METAPHASE Chromosomes line up at metaphase plate. Each chromatid now faces the pole opposite that of its sister chromatid. Figure 4 Mitosis and Cytokinesis. 1. Make the maternal chromosomes 10 beads and 4 beads long. The 10 bead chromosome will have 5 red beads on each side of the centromere and the 4 bead chromosome will have 2 red beads on each side of the cen tromere (Figure 3). 2. Repeat step 1 using blue instead for the paternal chromosomes. There should be a total of 4 chromosomes when you arc finished. 3. Make a centriole pair by attaching 2 beads of a different color to each other. 4. Using a very long piece of string, make a cell and place your bead structures inside. Assume that a nucleus is present, but we will not diagram one during this exercise. You are going to proceed through the stages of the cell cycle. 5. S phase and Gi phase Replication of the chromosomes occurs in the S phase and the centriolcs finish repli cation in the G2 phase. Make 4 more chromosomes just like your originals and also make another ccnlriolc pair. 6. Prophase I The centrosomes migrate to the opposite ends of the cell as the spindle apparatus starts to form between them. The chromosomes shorten and thicken and the nuclear envelope disassembles. Using additional pieces of string show the spindle. Each chromosome locates its homologous partner and forms an attachment call the synaptonemal complex. Once the homologous chromosomes find each other, they are now termed tetrads because there are now four sister chromatids in very close proximity to one another. The sister chromatid of one homologous chromosome that is closest to the sister chromatid of the other homologous chromosomes undergoes an exchange of chromosome pieces with each other in a process called crossing over. This process allows for the genetic exchange or recombination of genetic information in a form that was different than the original chromosomes. The other sister chromatids of the tetrad cannot undergo crossing over because they arc (oo far apart for this process to occur. Simulate this occurrence with your blue and red beads.

5 75 separating chromatids ANAPHASE Separation. Sister chromatids are moved to opposite poles in the cell. Each chromatid is now a full-fledged chromosome. TELOPHASE AND CYTOKINESIS Exit from mitosis. Chromosomes decondense, the mitotic spindle breaks down, and nuclear envelopes form around the two separate complements of chromosomes. Meanwhile, a cleavage furrow begins to form near the middle of the cell. COMPLETION OF CYTOKINESIS One cell becomes two. The cell membrane pinches together completely, the membranes on either side fuse together, and the one cell becomes two. These two cells now enter the G, phase of interphase. Figure 4 (continued) Reproduced from A Laboratory Guide to the Natural World, (2003). by permission of Carolina Bio Supply Company. 7. Prometaphase I The spindle apparatus is completely formed with the centrosomes placed opposite of each other along the width of the cell. This phase is where the spindle fibers attach to the kinetochores of the chro mosomes and start to move the chromosomes toward the metaphase plate (equatorial plate). 8. Metaphase I The chromosomes line up along the metaphase plate in pairs. The kinetochores of the outside sister chromatids are attached to their respective centrosomes. The inside sister chromatids are still attached at chiasma which are the result of crossing over. When all tetrads are arranged along the metaphase plate, then the signal is given for anaphase I to begin. This stage also leads to genetic diversity. Each tetrad lines up along the metaphase plate independently of the next tetrad of another chromosome. This process is completely ran dom and leads to the maternal chromosome orienting to one centrosome on one side of the metaphase plate while the paternal chromosome of the tetrad orients to the opposite centrosome on the opposite side of the metaphase. This process is called independent assortment. Practice different orientations with your chromo somes. Check with your classmates to see how many different combinations were created. 9. Anaphase I Immediately after the chromosomes line up in metaphase the spindle fibers will pull the homolo gous chromosomes apart from each other toward their respective centrosome. 10. Telophase I Once the homologous chromosomes reach the centrosomes and stop moving then this phase will begin. Cytokinesis begins at this time and the spindle begins to disintegrate. Toward the end of telophase the nuclear envelope will reappear. The chromosome number has been reduced by half at the end of telophase I. One set of chromosomes is now in one daughter cell and the other set of chromosomes is in the other daugh ter cell. The cells are now haploid, but they still contain the replicated chromosomes that have to be separated. *The cell does not replicate again as it is still in the M phase of the cell cycle. 11. Prophase II The centrosomes migrate to opposite ends of the cell as the spindle apparatus starts to form between them. The chromosomes shorten and thicken and the nuclear envelope disassembles. Using additional pieces of string show the spindle.

6 76 MEIOSISI Diploid END OF INTERPHASE DNA has already duplicated PROPHASEI Homologous chromosomes link as they condense, forming tetrads. METAPHASEI Mlcrotubules move homologous chromosomes to metaphase plate. ANAPHASEI Microtubules separate homologous chromosomes (sister chromatids remain together). Recombination occurs. Independent assortment occurs. RECOMBINATION Two very important sources of genetic \ variation Exchange of parts of non-sister chromatids. dup.icated maternal chromosome chromosome tetrad sister chromatids non-sister chromatids Reproduced from A laboratory Guide to the Natural World, (2003). by permission of Prentice-Hall, Inc. Figure 5. Meiosis, Crossing Over, and Independent Assortment.

7 77 MEIOSISII Haploid TELOPHASEI PROPHASE II METAPHASE II Two haploid daughter cells result from cytokinesis. (Brief) Sister chromatids line up at new metaphase plate. ANAPHASE II TELOPHASE II Sister chromatids separate. Four haploid cells result. INDEPENDENT ASSORTMENT Random alignment of maternal/paternal chromosomes at the metaphase plate. METAPHASE II TELOPHASEI METAPHASEI In the sequence above, homologous chromosomes lined up this way in metaphase I but they could have lined up this way, yielding a different outcome. Compare these to cells above Figure 5. (continued)

8 Prometaphase II The spindle apparatus is completely formed with the ccntrosomes placed opposite of each other along the width of the cell. This phase is where the spindle fibers attach to the kinetochores of the chro mosomes and start to move the chromosomes toward the metaphase plate. 13. Metaphase II Each chromosome lines up along the metaphase plate with one kinetochorc facing one pole while the other kinetochore faces the opposite pole. This creates a tension that allows for the signal to be given that indicates the start of anaphase. This is identical to mitosis because both sister chromatids are attached to oppo site ccntrosomes. 14. Anaphase II Immediately after the chromosomes line up in metaphase the spindle libers will pull the sister chromatids apart from each other toward their respective centrosome. They arc now considered chromosomes again after the sister chromatids have been split apart. 15. Tclophase II Once the chromosomes reach the ccntrosomes and stop moving then this phase will begin. Cytoki nesis begins at this time and the spindle begins to disintegrate. Toward the end of telophase the nuclear enve lope will reappear. Your resulting daughter cells will de different from each other as well as different from the parent cell. The chromosome number has now been reduced to half that of the beginning parent cell. *The comparisons between mitosis and meiosis are shown in Figure 6.

9 79 2N MITOSIS 2N 2N N N MEIOSIS Pearson Custom Publishing COMPARISON OF MITOSIS AND MEIOSIS Figure 6. Comparison of Meiosis and Mitosis.

10 80 Activity C. Plant and Animal Mitosis In this exercise you will look at two slides, onion root tip (plant) and a very early animal embryo. In each of these slides find prophase, metaphase, anaphase and telophase. Each of these viewed stages need to be sketched on the answer sheet. Both slides should be viewed on the high dry objective. The plant cells undergoing mitosis will be found closer to the rounded tip of the root in an area called the meristematic region. The animal slide will have several embryos each of which will have representative phases of mitosis. v.'.wtfjsfflt 1 ' ' " '.- V;'- Figure 7 Mitosis in Onion Root Tip Figure 8 Mitosis in a Fish Embryo. I'sod by permission of Carolina Biological Supply Company.

11 81 Cell Division Answer Sheet Name. Section. Date 1. Name two ways that genetic diversity is accomplished during cell division. At what cell division stages do these events occur? 2. How do the chromosomes of an anaphase cell of mitosis differ from those of an anaphase cell of meiosis I? 3. What major event happens just before prophase I but does not occur before prophase II? 4. Which results in the change in chromosome number from diploid to haploid, meiosis I or meiosis II? 5. Do the two things mentioned in question 1 happen in the stages of mitosis? Why or why not? Sketch the plant cells that are undergoing each stage of mitosis (prophase, metaphase, anaphase, telophase) and interphase. Label the determining characteristics of each stage.