Objectives:1. To begin to understand Reproduction and Life Cycles.2. To get hands-on experience with mitosis.3. To get hands-on experience with meiosis.4. To compare experimental vs. observational studies.

As we discussed in class, biological growth is a multi-dimensional phenomena. Basically, the basic unit of life, the cell, must duplicate itself. One "mother" cell must become two "daughter" cells, through the process of Cell Division, mostly MITOSIS. This is a completely asexual event and involves replication of the nucleus and its constituents and dividing up of the cytoplasm, to make two identical cells.....

.............. ..........MEIOSIS, on the other

hand, is involved only in sexual reproduction, and is the main source of genetic variation. In meiosis, cells with a full compliment of chromosomes divide to yield cells with one-half as many chromosomes. These cells become the "Egg" and the "Sperm" that unite later in a process known as FERTILIZATION.

Mitosis and meiosis also allow us to see science from a non-experimental perspective. Sometimes, science just records what it observes. Cells in actively growing regions of plants (meristematic regions), such as roots and shoots tips, have cells that are easily stained to show the process of mitosis. From these cells we can see cells actively dividing and at different steps in the process. The process of mitosis is ubiquitous in all diploid organisms. Often times, original, detailed descriptions of observations of processes have been the basis of future, experimental work. The observational descriptions we make today about mitosis and meiosis will allow us to develop hypothesis about genetic relationships in the next few labs. Actually it will be very important that you do not lose track of these basic principles of heredity for all the kernels of corn that you count.

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Lab #7: Mitosis and Meiosis

II Forming HypothesesRecall the Experiment Cycle or Scientific Process:

1. Make observations about the natural world.2. Ask questions about those observations.3. Formulate a reasonable testable hypothesis to explain observations.4. Create, execute, and replicate experiments testing the hypothesis and

generating results.5. Analyze results and draw inferences. This stimulates further inquiry. The

cycle begins anew.

Observation: Descriptions of the steps in mitosis and meisos.

Question1: What is the process by which organisms grow and replace cells?

Question 2: What is the process by which organisms produce gametes or sexually reproducing cells?

Hypothesis 1: The process of mitosis is composed of five steps that replicate the chromosomes and then divides the genetic material equally between two daughter cells.

Hypothesis 2: The two stages of meiosis replicate and reduce the number of chromosomes to produce four cells.

Experiment: Observing and describing the process of mitosis and meiosis in several sections of onion cells (mitosis) and a fungus (meiosis).

Results: Record your diagrams and descriptions in your lab book

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Lab #7: Mitosis and Meiosis

I. Background Material

A. Cell CycleAs a plant grows, new cells and nuclei arise from preexisting cells, Mitosis.

Mitosis occurs predominantly in meristematic regions of the plant: the root and stem tips, vascular and cork cambium, and in organs in early stages of growth. The two stages of mitosis are generally recognized as:

a. Mitosis - division of the nucleus into two nuclei;b. Cytokinesis - division of the cytoplasm and building of a new cell

wall laid down between the new cells.

In actively dividing cells, the term Cell Cycle is used to describe the life history of the cell, as it is made during mitosis, enlarges and develops, and then itself divides into two new cells. In other words, repeated cellular divisions are separated by periods of time during which growth and preparation for the next division occur.

During the "S" or synthesis phase of the Cell Cycle DNA is replicated. Since DNA is the "stuff" of which CHROMOSOMES are made, this replication results in the doubling of all chromosomes. This is necessary because during mitosis the chromosomes of one nucleus are divided between the two new nuclei.

PART 1 - "Mitosis in African Blood Lily".This part of the lab will be devoted to looking at the Mitotic phase of the

cell cycle. Although mitosis is often divided into phases, this only a pedagogical (teaching) tool. Prophase, prometaphase, metaphase, anaphase, and telophase are merely markers or frozen images (freeze frames) in a CONTINUOUS PROCESS. ................... To underscore this CONCEPT (mitosis is a continuous process) watch the short film of actual mitosis in Haemanthus.

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PART 2 - MITOSIS IN ONION (Allium cepa) ROOT TIPSObtain a prepared slide of onion root tips in longitudinal section. Under

low power, note the location of the apical meristem in relationship to cap and more mature regions. Under higher power try to find cells in various stages of mitosis.

Now you are going to look at fresh tissue squashes of onion roots to find your own mitotic stages. Get a fresh onion root from the container and observe it under the dissecting 'scope. Next, using a new razor blade, cut away and save the first 1 millimeter of the root tip containing the apical meristem. Place the tip on a glass slide and add a few drops of acid-alcohol. The acid-alcohol dissolves the pectin that holds the root cells together. Add more acid-alcohol, if necessary, to keep the mass of tissue moist.

After 5- 10 minutes, blot away the excess liquid and add a single drop of aceto-carmine stain and work this into the tissue using two probes. This takes time and patience, but soon the tissue will be a mass of bits. Finally, place a glass cover-slip over the cells and squash with a medium downward pressure, as directed by your TA.

Observe your squash under the microscope and search carefully for a region of mitotic activity. Again, this takes much patience, work and luck. Draw what you see in your lab books.

Find a region filled with actively dividing cells. Count and record the number of cells in each phase. What do the numbers mean for mitosis and the cell cycle?

Using the books available in lab, look at as series of pictures of the Phases of Mitosis. What is happening to chromosomes during the process of Mitosis?

Interphase:Prophase:Prometaphase:Metaphase:Anaphase:Telophase:

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PART 3 THE CIRCLE OF LIFE - MEIOSIS AND FERTILIZATIONTwo critical events occur in the life history of all sexually reproducing

organisms: meiosis and fertilization. During the interphase preceding meiosis, the chromosomal material; (DNA) is replicated. Then, during meiosis, the nucleus undergoes two divisions, one of which is a reduction division. By a precise mechanism, meiosis produces four daughter nuclei, each with one-half the number of chromosomes, and thus one-half as much DNA, as the parent nucleus. Whereas the parent nucleus is diploid (2n), each of the daughter nuclei is haploid (n). In diploid cells, the chromosomes are present in matched pairs called homologous chromosomes. Each parent contributes one member of each pair during sexual reproduction. In the reduction division of meiosis, the pairs of homologous chromosomes are separated. Haploid cells, therefore, contain only one member of each homologous pair of chromosomes. See your text for details.

Look at as series of pictures of the Phases of Meiosis in the various biology books in the lab. What is happening to chromosomes during the process of Meiosis?

In the lab various colors of clay are available. Demonstrate the steps of meiosis to your TA, using two sets of homologous chromosomes, moving the clay models through the phases of Meiosis I and Meiosis II, first without crossing over and secondly with crossing over. Be especially attentive to Metaphase I, crossing over, and independent assortment. (Use different colors of clay to represent the two sets of homologous chromosomes).

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II. CROSSING OVER In this part of lab you will look at genetic segregation and the frequency of crossing over in a fungus. By understanding the process of crossing over, you can tie together the principles of DNA structure with inheritance of characters (Mendelian genetics).

GENETIC SEGREGATION AND CROSSING OVERIn meiosis, genetic recombination may occur as a result of the exchange of genetic material between homologous chromosomes during the process of crossing over. Crossing over occurs during prophase I, when homologous chromosomes synapse. While they are joined in this complex, nonsister chromatids may break at corresponding points and exchange parts. A point at which they temporarily joined as a result of this exchange is called a chiasma.

Figure 1

Sordaria fimicola is a saprotrophic fungus that occurs on dung and decaying plant matter. It belongs to the class Ascomycetes, a group characterized by saclike sporangia called asci (singular, ascus), each of which, at maturity, contains eight linearly arranged haploid (n) ascospores (see figure 1). The asci (about 20) are grouped together within a structure called the perithecium. It is the dark brown perithecium on the agar plate that you can observe with the naked eye. When the ascospores within a given ascus are mature, the ascus elongates and penetrates into the neck of the perithecium. Upon reaching the top of the neck, the tip of the ascus breaks open and spore discharge occurs. The spent ascus quickly shrivels up, and another one begins to elongate.

Genetics of Sordaria fimicola

Sordaria fimicola spends most of its life as a haploid mycelium, a mass of cells arranged in filaments. When conditions are favorable, cells of filaments from two different mating types fuse. Ultimately, the nuclei fuse and 2n

zygotes are produced (see figure 2). Each 2n zygote undergoes meiosis, and the resulting cells (ascospores) remain aligned. The position of an ascospore within the ascus depends on the orientation of separating chromosomes on the equatorial plane of meiosis I. After meiosis, each resulting ascospore divides once by mitosis, resulting in eight ascospores per ascus. This unique sequence of events means that it is easy to detect the occurrence of crossing over involving chromatids carrying alleles that encode for color of spores and mycelia.

If two mating types of Sordaria, one with black spores and the other with tan spores, are grown on the same petri dish, mycelia from the two may grow together, and certain cells may fuse. Nuclei from two fused cells then fuse, and the resulting zygote contains one chromosome carrying the allele for black spores and another carrying the allele for tan spores. After meiosis I and II take place, one mitosis follows, and the result is eight ascospores in one ascus: four black spores and four tan spores. If no crossover events occur, the two genes will segregate during meiosis I and produced a 4:4 arrangement of ascospores.  If a  crossover event does occur, the two genes will not segregate until meiosis II resulting in a 2:2:2:2 or 2:4:2 sequence of ascospores (Figure 3).  

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Map Distance: The genes on a chromosome are situated at specific distances from the centromere. For any given gene, that distance remains constant. In the case of a gene located close to the centromere, few if any, crossovers are to be expected in the gene-to-centromere interval. If the gene is farther removed from the centromere, a greater number of crossovers are to be expected between it and the centromere. In other words, the greater the distance of the gene from the centromere, the greater the frequency of crossovers in the gene-to-centromere interval. Since the frequency of crossovers in the gene-to-centromere interval is a function of the distance of the gene to centromere, the crossover frequency can be used to determine how far from the centromere a gene is located.

Map distances for haploid organisms can be calculated. Map distances are estimated on the basis of the frequency of second division (MII) segregation as follows. First, the total number of second division (MII) segregation asci are determine. This number is them divided by the total number of bicolored asci that were counted. The resulting number is multiplied by 100 to obtain the percentage of second division segregation. This percentage of crossovers must be divided in half in order to obtain the map distance between the gene locus and the centromere. Division by 2 is necessary because only two of the four chromatids of the tetrad are involved in crossing over. The value obtained by dividing % MII by 2 (the relative distance between the locus and the centromere) is expressed in arbitrary units called map units.

Analysis of Hybrid Perithecia1. From your instructor obtain the following cross plate:

Wild-type (+) by tan spored mutant (t)

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2. Working under a dissecting microscope and using a dissecting needle, transfer four or five perithecia from the cross plate into a drop of distilled water on a clean glass slide. Select perithecia from those regions of the cross plate where the mycelia of the two strains are in contact. Cover the perithecia with a coverslip.

3. Tap gently on the coverslip with the blunt end of a dissecting needle to break open the perithecia and release the asci. Do not move the coverslip.

4. Examine your preparation under the compound microscope for the presence of hybrid perithecia. If no hybrid perithecia are present, make a new slide.

5. If hybrid perithecia are present, remove the slide from the microscope. Place a piece of blotting paper over the coverslip and, while holding the coverslip in position with one hand, apply gentle pressure to the coverslip with the thumb of your free hand.

6. Using the compound microscope, locate a cluster (rosette) of hybrid asci and count the number of first and second division segregation asci (MI and MII). Record the data in Table 1.

7. When all of the asci in a given cluster have been counted, locate another cluster of hybrid asci and again count the number of first and second division segregation asci, entering the data in Table 1. When all of the asci on the slide have been counted, prepare a new slide and again count the hybrid asci. Count 200 bi-colored asci.

8. Using your data, determine the percent of second division segregation of asci

(% MII).

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Sordaria Laboratory Worksheet

Number of Meiosis I Asci 4:4

Number of Meiosis II Asci 2:2:2:2 or 2:4:2

Total Number of Asci

% Meiosis II Asci / Total # asci counted)

Gene to Centromere Distance(% Meiosis II / 2)

1. How many different arrangements did you find? __________________

2. What were the different arrangements? To answer this question list the black spores as + and the tan (mutant) spores as t. For example, no crossing over would be recorded as ++++ t t t t

3. How do you explain the perithecia in which the asci contained either eight black or eight tan ascospores?

4. Were any other ascospore arrangements observed other than the ones previously mentioned? If so, how might these be explained?

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