Computer

Mitosis and Meiosis

Part I Mitosis

It was discovered in 1858, by Rudolf Virchow, that new cells can only arise from previously existing cells. This is done in two ways: mitosis and meiosis. Somatic (body) cells divide exclusively by mitosis followed by cytokinesis, while germ cells produce gametes by the process of meiosis. Plant cells grow by enlargement, essentially by taking up water. When they reach a certain size, they divide, forming two identical daughter cells. The various parts of the cell are divided in such a way that the new daughter cell is identical to the parent cell. Strictly speaking, mitosis implies only the division of the nucleus, and is therefore distinct from cell division, in which the cytoplasm is divided. In most organisms, cells divide by ingrowth of the cell wall, if present, and the contraction of the cell membrane, a process that cuts through the spindle fibers. In land plants (bryophytes and vascular plants) and a few algae, cell division takes place by the formation of a cell plate. Small droplets appear across the equatorial plate of the cell and gradually fuse, forming a disc that grows outward until it reaches the wall of the dividing cell, which completes the separation of the two daughter cells. The DNA of prokaryotes is simply replicated before division. In eukaryotes, however, the hereditary material is part of their complex chromosomes. Equal division of this material requires a more complex method by which the chromosomes are replicated, separated, and apportioned precisely between the daughter cells. Mitosis, or nuclear division, ensures the equal division of the nuclear material between the daughter cells in eukaryotic organisms. During mitosis the chromosomes condense, and move to the center of the cell where they fully contract. They then split longitudinally into two identical halves that appear to be pulled to opposite poles of the cell by a series of microtubules. In these two genetically identical groups, the coiling of the chromosomes relaxes again, and they are reconstituted into the nuclei of the two daughter cells. It is a continuous process that can be divided into five major phases: interphase, prophase, metaphase, anaphase, and telophase. Interphase: The chromatin, if visible at all, can only be seen as small grains or threads. Interphase is generally considered to be a resting phase. However, the cell is replicating the genetic material, preparing for mitosis. Prophase: The beginning of mitosis is illustrated by the chromosomes gradually becoming visible. They start out as elongated threads that shorten and thicken. Chromosomes become more condensed and undergo spiral contractions, like a thin wire being turned into a coiled spring. This coiling involves the entire DNAprotein complex. Each chromosome is composed of two longitudinal halves, called chromatids, joined in a narrow area known as the centromere, where the chromatids are not coiled. The centromere, located on each chromosome, divides the chromosomes into two arms of varying lengths. As prophase progresses, the nucleoli grow smaller and finally disappear. Shortly after, in most cell types, the nuclear envelope breaks down, putting the contracted chromosomes into direct contact with the cytoplasm; this marks the end of prophase.

Advanced Biology with Vernier

3-1

Computer 3 Metaphase: The chromosomes, still doubled, become arranged so that each centromere is on the equatorial region of the spindle. Each chromosome is attracted to the spindle fibers by its centromere; often the arms of the chromosome point toward one of the two poles. Some of the spindle fibers pass from one pole to the other and have no chromosome attached.

Anaphase: The chromatids separate from one another and become individual chromosomes. First, the centromere divides and the two daughter chromosomes move away from the equator toward opposite poles. Their centromeres, which are still attached to the spindle fiber, move first, and the arms drag behind. The two daughter chromosomes pull apart; the tips of the longer arms separate last. The spindle fibers attached to the chromosomes shorten as the chromatids divide and the chromosomes separate. The fibers appear to move, but in fact the microtubules are continuously formed at one end of the spindle fiber and disassembled at the other. In the process, it appears as if the spindle fibers were tugging the chromosomes toward the poles by their centromeres. By the end of anaphase, the two identical sets of chromosomes have separated and moved to opposite poles. Telophase: The separation is made final; the nuclear envelopes are organized around the two identical sets of chromosomes. The spindle apparatus disappears. Nucleoli also reform at this time. The chromosomes become increasingly indistinct, uncoiling to become slender threads again.

Cytokinesis: As mitosis ends, cytokinesis begins, resulting in the formation of two daughter cells. The cleaved membrane slowly draws together, forming a narrow bridge, then separates the cell into two daughter cells. The cells now enter interphase.

Mitosis ends when the processes are complete and the chromosomes have once more disappeared from view. The two daughter cells enter interphase. The two daughter nuclei produced are identical to one another and to the nucleus that divided to produce them. In order to investigate the process of mitosis, plant and animal tissues where cells are dividing rapidly must be examined. In animals, the most rapidly growing and dividing tissues are found in the embryonic stages of development. Although most animal tissues continue to undergo mitosis throughout the life cycle of the organism, they do so very slowly when compared to their embryos. Some animal cells, like most plant tissues, rarely replicate after the organism reaches maturity. In plants, these tissues are primarily found in the tips of stems and roots. The root tip plants are exceptionally good places to look for cells undergoing mitosis. Plant root tips consist of several different zones where various developmental and functional processes of the root are performed.

3-2

Advanced Biology with Vernier

Mitosis and Meiosis The primary region for the formation of new cells is the apical meristem. The root cap offers protection for the rest of the root, the region of elongation is the area where the bulk of cell growth occurs, and the region of maturation is where tissue differentiation occurs.

Part II Meiosis
Sexual reproduction provides a mechanism to produce genetic variation, as the genes of two different individuals are arranged in various ways. This requires a reduction in the chromosome number of the parent cell, normally diploid, to half that, or haploid, in somatic cells. The type of cell division resulting in half the chromosome number of the parent cell is called meiosis. In meiosis, a germ cell divides into four haploid gametes. When two gametes egg and spermcombine during fertilization, forming a zygote, the diploid chromosome number is restored. Meiosis consists of one DNA replication and two nuclear divisions, meiosis I and II. This results in the formation of four daughter cells, each with only half the number of chromosomes as the parent. Genetic variability is further increased by a process called crossing over. In the early stages of meiosis, the homologous pairs of chromosomes move close together in such a way that all four chromatids are entwined, forming a tetrad. This process, known as synapsis, allows for the exchange of chromosome sections between the homologous pairs. The example that will be used in the investigation is Sordaria fimicola, an ascomycete fungus that is haploid for the bulk of its life cycle, including the individual fungal filaments, called hyphae, which normally exist in a mass called a mycelium representing the body of the fungus; and the ascospores, from which mycelia develop. The only diploid portion of the life cycle of S. fimicola occurs when the nuclei of specialized hyphae come together. These hyphae, which belong to different strains of the species, fuse to form a zygote. This zygote then undergoes meiosis to produce the haploid ascospores, yielding four haploid nuclei contained in a sac called an ascus. After meiosis, the four nuclei undergo mitosis, resulting in an ascus containing eight haploid ascospores. Many asci form inside a fruiting body called a perithecium.

Advanced Biology with Vernier

3-3

Computer 3 One type of genetic variability in S. fimicola is the color of the ascospores. Most strains are the dark brown, wild-type ascospores, although there are variants. Certain strains have tan or gray ascospores. A tan ascospore strain mated with the wild-type variety produces a series of perithecia containing asci with four tan and four wild-type ascospores each. How these ascospores are arranged within the ascus is a direct representation of whether or not crossing over has occurred between the centromere and the site for the gene for ascospore color. If no crossing over has occurred, the ascospores will be arranged in a 4:4 manner. If crossing over has occurred, they will occur in a 2:4:2 or 2:2:2:2 manner. By observing the ascospore arrangement, the percentage of asci exhibiting crossover can be determined. This frequency appears to be affected largely by the distance from the gene to the centromere. From the crossover frequency, the distance in map units from the gene for ascospore color, and the chromosome centromere, can be calculated.

OBJECTIVES
In this experiment, you will Examine and compare the phases of mitosis in animal and plants cells. Determine the relative time cells spend in each phase of mitosis. Prepare microscope slides of mitotic cells using onion Allium root tips. Follow the processes of mitosis and meiosis in the life cycle of Sordaria. Examine the arrangement of Sordaria ascospore microscopically to determine the frequency of crossing over. Calculate the distance, in map units, between a specific gene and the chromosome centromere.

3-4

Advanced Biology with Vernier

Mitosis and Meiosis

Part I Mitosis MATERIALS

Materials for Parts A and B

whitefish mitosis slide onion mitosis slide compound microscope

Materials for Part C

onion root tip hydrochloric acid 1M toluidine blue 0.5% compound microscope coverslip microscope slide

Bunsen burner clothespin or forceps paper towel pipet scalpel

PROCEDURE
Part A: Observing Mitosis in Plant and Animal Cells

1. Observe the prepared microscope slide of onion root tip mitosis, first at 100X, then 400X. Using the Plant Mitosis Chart as a guide, identify cells that represent each mitotic phase. 2. In the Analysis section, draw each phase of plant cell mitosis that you see. Write a brief description of each phase below each drawing. 3. Observe the prepared microscope slide of whitefish blastula. Using the Animal Mitosis Chart as a guide, identify each phase of animal cell mitosis. 4. In the Analysis section, draw each phase of plant or animal cell mitosis that you see. Write a brief description of each phase below each drawing.
Part B: Relative Lengths of Phases of Mitosis

5. Examine at least three fields of view of the apical meristem of the onion root tip at 400X. In each view, count the number of cells in the various stages of mitosis. Record this data in Table 1. 6. Calculate the total number of cells counted and the percentage of total cells counted for each stage of mitosis. Record this data in Table 1. Record the percentages in Table 2, as well. 7. Assuming that it takes an average of 24 hours (1,440 minutes) for onion root tip cells to complete the cell cycle, calculate the amount of time cells spent in each phase of the cycle. Use the formula provided below. Enter your results in Table 1. Percent of Cells in Phase 1,440 minutes = _________ minutes cell spent in phase

Advanced Biology with Vernier

3-5

Computer 3
Part C: Preparation of an Onion Root Tip Squash

8. Wrap a fresh onion set with paper toweling and place it in a plastic sandwich bag. Add 10 mL of water before sealing the bag. Note: The set used must have root primordia present or it will not produce root tips. The root tips will grow within 36 to 72 hours. Common onions have a mitotic cycle of approximately 20 hours. 9. Place the plastic bag in a box or dark place until the root tips have grown to a length of about 4 to 5 mm. It is important that the onion grows in the dark to ensure that it produces roots rather than shoots. 10. Remove the onion from the box approximately one half-hour before performing the experiment to expose the root tips to light. 11. Blot as much excess water from the root tips as possible. Any excess water on the slide will affect your results. Do not allow the root tips to dry out. 12. Using a scalpel or razor blade, cut off the end of one of the emergent root tips; the section should be approximately 1 to 2 mm long. Place the root tip on a clean microscope slide and apply one to two drops of HCl on the root tip. 13. Holding the slide with a clothespin or forceps, pass it through the flame of a Bunsen burner for several seconds. Note: Do not hold the slide directly over the flame. 14. Without harming the root tip, blot the specimen with a paper towel to remove the excess HCl. Note: You may wish to touch a corner of the paper towel to the edge of the root tip and allow the paper towel to wick up the solution. 15. Add one to two drops of 0.5% aqueous toluidine blue stain to the root tip. Note: Toluidine blue is a mild irritant, avoid direct physical contact with this stain. 16. Pass the slide through the flame of a Bunsen burner for one to two minutes. Let the slide stand and cool for one minute. Note: Do not hold the slide directly over the flame. 17. Remove excess stain with a paper towel in a motion similar to the one used in Step 14. 18. With a scalpel or razor blade, slice the root tip longitudinally down the middle into two mirror segments. 19.Add a drop of toluidine blue and cover the root tip with a coverslip. Using the scalpel handle or other blunt instrument, gently press down on the coverslip to squash and spread out the root tip. Blot off excess stain, if any, that may come out from under the coverslip. 20. View the slide under a microscope using the 10X objective. Locate the apical meristem. Examine the slide using the 40X objective. Locate cells in the various stages of mitosis. Sketch the nuclear regions of individual cells in the five different stages and describe key features of each in the Analysis section. Keep in mind that since the root tip has been squashed, the meristem may not be readily recognizable.

3-6

Advanced Biology with Vernier

Mitosis and Meiosis

Part II Meiosis MATERIALS

microscope slide coverslip inoculating loop sordaria demonstration cross plate (shared)

PROCEDURE
1. Place a drop of water on a clean slide with an inoculating loop. 2. With an inoculating loop, scrape several perithecia from the demonstration cross plate. Scrape the perithecia from the interface of two crossing strains close to the edge of the plate and place in the drop of water on the slide. Avoid picking up agar along with perithecia; it will interfere with results. 3. Cover the slide with a coverslip. Using a pencil eraser or other blunt instrument, gently press down on the coverslip to squash and spread out the perithecia. The pressure should be sufficient to squeeze the asci from the perithecia, but not enough to crush the asci themselves. Note: It may be helpful to slide the coverslip around on top of the sample, with slight pressure, to spread out the asci and make them easier to observe. Keep in mind, however, that applying too much pressure may rupture the asci, releasing the individual ascospores. 4. View the slide under a microscope at 100X. Locate the asci. You may wish to view the slide at 400X to determine the color of some ascospores. The slide preparation should show collapsed perithecia and asci clusters (rosettes), with mature ascospores in various arrangements. Immature ascospores will all be light colored. Since S. fimicola is homothallic, the preparation will show both hybrid and self-fertilized perithecia of both parental types. Hybrid perithecia, however, will not occur very far from the line of contact between the two varieties. Prepare three slides to get an adequate sampling of hybrids, if possible. 5. Count approximately 50 hybrid asci from at least three fields of view, preferably from different slides. Record the data in Table 2.

Advanced Biology with Vernier

3-7

Computer 3

ANALYSIS

Stage: ___________ Description: ______ _________________ _________________ _________________ _________________

Stage: ___________ Description: ______ _________________ _________________ _________________ _________________

Stage: ___________ Description: ______ _________________ _________________ _________________ _________________

Stage: ___________ Description: ______ _________________ _________________ _________________ _________________

Stage: ___________ Description: ______ _________________ _________________ _________________ _________________

3-8

Advanced Biology with Vernier

Mitosis and Meiosis

Table 1 Cells in each stage of mitosis Stage Interphase Prophase Metaphase Anaphase Telophase # of cells in Field 1 # of cells in Field 2 # of cells in Field 3 Total # of cells % of total # of cells Time of each stage (min)

Total number of cells counted __________

Table 2 Percentage of cells in each stage of mitosis Stage Interphase Prophase Metaphase Anaphase Telophase Total % of total # of cells

Table 3 Observed Sordaria Asci # of 4:4 # of crossover Total % asci showing crossover Gene distance from centromere

Using your data, determine the distance in map units from the gene for ascospore color to the chromosome centromere. Calculate the percentage of asci that showed crossover. This percentage crossover must be divided by two, since only half the ascospores in each hybrid ascus are the result of crossing over. Dropping the % symbol gives you the map distance from the gene to the centromere. Record the data in Table 3. % Crossover = # showing crossover 100% total counted Gene Distance from Centromere = % Crossover

Advanced Biology with Vernier

3-9

Computer 3

QUESTIONS

1. Referring to the percentage of total cells counted in each phase of mitosis, determine which phase takes the longest for the cell to complete, and explain why. Sketch a pie graph of the percentage of cells in each phase to illustrate. Be sure to title your graph and include a key. 2. What is the relationship between the processes of mitosis and cytokinesis? 3. Which of the following is significantly different between plant and animal cell mitosis? a. b. c. d. metaphase anaphase cytokinesis prophase

4. How does meiosis lead to genetic variability within a population? Use S. fimicola as an example. 5. Define the following terms. somatic cell germ cell chromatin centromere diploid haploid zygote 6. Create a Venn diagram showing at least two similarities and two differences between mitosis and meiosis.

3 - 10

Advanced Biology with Vernier

Mitosis and Meiosis

ANIMAL CELL MITOSIS

Interphase

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

Advanced Biology with Vernier

3 - 11

Computer 3

PLANT CELL MITOSIS

Interphase

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

3 - 12

Advanced Biology with Vernier