Mitosis and Meiosis

I. Time for Cell Replication Overview

To estimate the relative length of time that a cell spends in the various stages of cell replication, you will examine the meristematic region of a prepared slide of the onion root tip. The length of the cell cycle is approximately 24 hours for cells in actively dividing onion root tips.
It is hard to imagine that you can estimate how much time a cell spends in each phase of cell replication from a slide of dead cells. Yet this is precisely what you will do in this part of the lab. Since 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 percent of time each cell spends in each phase.

1. Observe every cell in one high power field of view and determine which phase of the cell cycle it is in. This is best done in pairs. The partner observing the slide calls out the phase of each cell while the other partner records. Then switch so the recorder becomes the observer and vice versa. Count at least two full fields of view. If you have not counted at least 200 cells, then count a third field of view.
2. Record your data in Table 1

Table 1
Field 1 Field 2 Field 3 Total Percent of total cells Time in each stage
Total cells:

3. Calculate the percentage of cells in each phase.

Consider that it takes, on average, 24 hours (or 1,440 minutes) for onion root-tip cells to complete the cell cycle. You can calculate the amount of time spent in each phase of the cell cycle from the percent of cells in that stage.

Percent of cells in stage X 1,440 minutes = minutes of cell cycle spent in stage

1. If your observations had not been restricted to the area of the root tip that is actively dividing, h6w would your results have been different?

2. Based on the data in Table 3.1, what can you infer about the relative length of time an onion root-tip cell spends in each stage of cell division?

II. Crossing Over during Melosis in Sordaria

Sordaria fimicola is an ascomycete fungus that can be used to demonstrate the results of crossing over during meiosis. Sordaria is a haploid organism for most of its life cycle. It becomes diploid only when the fusion of the mycelia of two different strains results in the fusion of the two different types of haploid nuclei to form a diploid nucleus. The diploid nucleus must then undergo meiosis to resume its haploid state.
Meiosis, followed by mitosis, in Sordaria results in the formation of eight haploid ascospores contained within a sac called an ascus (plural, asci). Many asci are contained within a fruiting body called a perithecium. When ascospores are mature the ascus ruptures, releasing the ascospores. Each ascospore can develop into a new haploid fungus. The life cycle of Sordaria fimicola is shown in Figure 1.

To observe crossing over in Sordaria, one must make hybrids between wild-type and mutant strains of Sordaria. Wild-type (+) Sordaria have black ascospores. One mutant strain has tan spores (tn). When mycelia of these two different strains come together and undergo meiosis, the asci that develop will contain four black ascospores and four tan ascospores. The arrangement of the spores directly reflects whether or not crossing over has occurred. In Figure 2, no crossing over has occurred. Figure 3 shows the results of crossing over between the centromere of the chromosome and the gene for ascospore color.

Figure 2

Two homologous chromosomes line up at metaphase I of meiosis. The two chromatids of one chromosome each carry the gene for tan spore color (tn) and the two chromatids of the other chromosome carry the gene for wild-type spore color (+).
The first meiotic division (MI) results in two cells each containing just one type of spore color gene (either tan or wild-type). Therefore, segregation of these genes has occurred at the first meiotic division (MI).
The second meiotic division (MII) results in four cells, each with the haploid number of chromosomes (lN).
A mitotic division simply duplicates these cells, resulting in 8 spores. They are arranged in the 4:4 pattern.

Figure 3

In this example, crossing over has occurred in the region between the gene for spore color and the centromere. The homologous chromosomes separate during meiosis I. This time, the MI results in two cells, each containing both genes (1 tan, 1 wild-type); therefore, the genes for spore color have not yet segregated.
Meiosis II (MII) results in segregation of the two types of genes for spore color.
A mitotic division results in 8 spores arranged in the 2:2:2:2 or 2:4:2 pattern. Any one of these spore arrangements would indicate that crossing over has occurred between the gene for spore coat color and the centromere.
Two strains of Sordaria (wild-type and tan mutant) have been inoculated on a plate of agar. Where the mycelia of the two strains meet (Figure 4), fruiting bodies called perithecia develop. Meiosis occurs within the perithecia during the forrnation of asci. Prepare a wet mount of some perithecia (the black dots in figure 4).

Figure 4

Gently press down on the coverslip so that the perithecia rupture but the ascospores remain in the asci. Using the 10x objective, view the slide and locate a group of hybrid asci (those containing both tan and black ascospores). Count at least 50 hybrid asci and enter your data in Table 2.

Table 2
Number of 4:4 asci Number of crossover asci Total asci % showing crossover / 2 Gene to centromere distance

The frequency of crossing over appears to be governed largely by the distance between genes, or in this case, between the gene for spore coat color and the centromere. The probability of a crossover occurring between two particular genes on the same chromosome (linked genes) increases as the distance between those genes becomes larger. The frequency of crossover, therefore, appears to be directly proportional to the distance between genes.
A map unit is an arbitrary unit of measure used to describe relative distances between linked genes. The number of map units between two genes or between a gene and the centromere is equal to the percentage of recombinants. Customary units cannot be used because we cannot directly visualize genes with the light microscope. However, due to the relationship between distance and crossover frequency, we may use the map unit.

Analysis of Results
1. Using the data in Table 2, determine the distance between the gene for spore color and the centromere. Calculate the percent of crossovers by dividing the number of crossover asci (2:2:2:2 or 2:4:2) by the total number of asci x 100. To calculate the map distance, divide the percentage of crossover asci by 2. The percentage of crossover asci is divided by 2 because only half of the spores in each ascus are the result of a crossover event (Figure 3). Record your results in Table 2.

2. Draw a pair of chromosomes in MI and MII, and show how you would get a 2:4:2 arrangement of ascospores by crossing over. (Hint: refer to Figure 3).