AP BIO Lab 3

AP BIO Lab 3: Mitosis and Meiosis

Part A: Mitosis

Background:
    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 DNA–protein 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.

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. 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.

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.

Materials:
whitefish mitosis slide
onion mitosis slide
compound microscope

Procedure:

Part 1: Observing Mitosis in Plant and Animal Cells

1. Observe the prepared microscope slide of onion root tip mitosis, first at with the 10X objective, then 40X objective. For convenience in discussion, biologists have described certain strages, or phases, of the continuous mitotic cell cycle. Identify one cell 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, first at with the 10X objective, then 40X objective. Identify each phase of animal cell mitosis.

4. In the Analysis section, draw each phase of animal cell mitosis that you see. Write a brief description of each phase below each drawing.

Part 2: Relative Lengths of Phases of Mitosis

It is hard to imagine that you can estimate how much time a cell spends in each phase of cell division from a slide of dead cells, yet this is precisely what you will do in this part of the lab. Sine you are working with a prepared slide, you cannot get any information about how long it takes a cell to divide. What you can determine is how many cells are in each phase. From this, you can infer the percentage of time each cell spends in each phase.

5. Examine at least three fields (~200 cells) of view of the apical meristem of the onion root tip with the 40X objective. This is best done in pairs. In each view, the partner observing the slide counts and calls out the number of cells in the various stages of mitosis and the other partner records. With each new view, the roles switch and the recorder becomes the observer. 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.

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

Data:

Table 1

	Number of Cells				Percent of Total Cells Counted	Time in Each Stage
	Field 1	Field 2	Field 3	Total
Interphase
Prophase
Metaphase
Anaphase
Telophase

Total Cells Counted________________

Analysis:

Part 1:
Drawing and brief description of cells that represent of each mitotic phase.

Interphase Prophase Metaphase Anaphase Telophase

1. Explain how mitosis leads to two daughter cells, each of which is diploid and genetically identical to the original cell. What activities are going on in the cell during interphase?

2. How does mitosis differ in plant and animal cells? How does plant mitosis accommodate a rigid, inflexible cell wall?

3. What is the role of the centrosome ( the area surrounding the centrioles)? Is it necessary for mitosis? Defend your answer.

Part 2:
1. If your observations had not been restricted to the area of the root tip that is actively dividing, how would your results have been different?

2. Based on the data in the Table, what can you infer about the relative length of time an onion root tip cell spends in each stage of cell division?

3. Draw and label a pie chart of the onion root tip cell cycle using the data from Table.

Title: ____________________

Part B: Meiosis

Background
    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 sperm—combine 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. 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
• Simulate mitosis, meiosis and chromosome cross overs using pipe cleaners.
• Examine the arrangement of Sordaria ascospore microscopically to determine then frequency of crossing over.
• Calculate the distance, in map units, between a specific gene and the chromosome centromere.

Procedure:

Part 1: Simulating Mitosis
1. Take pipe cleaners out of the zip lock bags and pair each one up with another pipe cleaner of same size and color. Put one pipe cleaner from each pair and one bean back into the zip lock bag. Record the number of chromosomes your parent cell has in the table. You are now ready to start the simulation.
2. Simulate each phase of Mitosis using the pipe cleaners. Once you have set up your supplies to show each phase, check with teacher that you did necessary steps before moving on to next phase.
3. Fill out the rest of Mitosis column of data table.

Part 2:Simulating Meiosis
1. Take pipe cleaners out of the zip lock bags and pair each one up with another pipe cleaner of same size and color. Put one pipe cleaner from each pair and one bean back into the zip lock bag. Record the number of chromosomes your parent cell has in the table. You are now ready to start the simulation.
2. For Interphase I, do same as in Mitosis.
3. For Prophase I, where homologous chromosomes (pair of same sized chromosomes of different colors-one that comes from each parent) come together and synapse along their entire length. This pairing of homologous chromosomes represents the first big difference between mitosis and meiosis. A tetrad, consisting of four chromatids, is formed.
3. For Metaphase I, line each homologous pair up in the middle of the cell.
4. For Anaphase I, the homologous chromosomes separate and are "pulled" to opposite sides of the cell. Chromosome number is therefore reduced.
5. For Telophase I, place each chromosome from the pair at opposite sides of the cell. Formation of a nuclear envelope and division of the cytoplasm often occur at this time to produce two cells, but this is not always the case. Notice that each chromosome within the two daughter cells still consists of two chromatids.
6. For Interphase II, the amount of time spent "at rest" following Telophase I depends on the type of organism, the formation of new nuclear envelopes, and the degree of chromosomal uncoiling. Because Interphase II does not necessarily resemble interphase I. DNA replication does not occur during Interphase II. This is the end of Meiosis I.
7. Meiosis II starts with Prophase II, no DNA replication occurs. Replicated centrioles separate and move to opposite sides of the chromosome groups.
8. For Metaphase II, orient the chromosomes so that they are centered in the middle of each daughter cell.
9. For Anaphase II, the centromere regions of the chromatids now separate. Now that each chromatid has its own visibly separate centromere region, it can be called a chromosome.
10. For Telophase II, place the chromosomes at opposite sides of the dividing cell. At this time a nuclear envelope forms and the cytoplasm divides. Fill in the Table for Part 1 and 2 for Meiosis.
11. Now start over with the simulation but this time when you get to Prophase I, we are going to simulate crossing over. Crossing over can be simulated but cutting one end of one of the sister chromatids for each homologous pair and switching them by reconnecting with twists when they are lined up. Then continue with Meiosis I and II like first time around.

Part 3:Exploring Sordaria to determine frequency of crossing over and distance between a gene and the centromere.
1. Look at Sordaria slides on Handout and count approximately 50 hybrid asci from at least three fields of view, preferably from different slides. Record the data in Table for Part 3.
2. To find the % of spores (asci) crossing over take the number of hybrids (2:2:2:2 or 2:4:2) and divide it by the number of total Asci and multiply by 100.
3. Take that % and divide it by 2. Place answer in Table for Part 3. This will give you the amount of map units the gene that controls color is from the centromere. A map unit is an arbitrary unit used to tell the distance from one gene to another gene. It isn’t a conventional unit because it can’t be seen using a light microscope.

Data Tables:

Part 1 and 2:

	Mitosis	Meiosis
Chromosome Number of Parent Cell
Number of DNA Replications
Number of Divisions
Number of Daughter Cells
Chromosome Number of Daughter Cells
Purpose/Function

Part 3:

Number of 4:4 Asci	Number of Asci Showing Crossover	Total Asci	% Asci Showing Crossover Divided by 2	Gene to Centromere Distance (map units)

Analysis Questions:

Part 1 and 2:

1. List three differences between the events of mitosis and meiosis.

2. How are Meiosis I and Meiosis II different?

3. How are oogenesis and spermatogenesis different?

4. Why is meiosis important for sexual reproduction?