Mitosis and Meiosis LabNotes From the teacher

Day 1 (to be done at home): Review the lab through PART A (Online Mitosis Practice with Onion Root Tip and Mitosis Drawing). Complete the pre-lab for Part A:

o Title and date of the lab (remember to add this lab to your table of contents). o Pre-Lab Questions you need to number the question, rewrite the question, and then answer it

for full credit (you will only be doing prelab questions once…they encompass the whole lab)o Write a sub-title: PART A Online Mitosis Practice with Onion Root Tip and Mitosis Drawingo Purpose 1-2 sentences describing the overall goal of PART A of the experiment; use

complete sentenceso Lab Procedure Write a procedure for PART A. Use your own words and put in numbered

steps. o Data Tables Either recopy or cut out and paste Tables 1.1 and 1.2 into your lab notebook.

Complete PART A of the lab. You will need a computer to do this. Complete the Analysis Questions for PART A of the lab. Complete the prelab for Part B (Time Spent in Each Phase of the Cell Cycle)

o Subtitle Part B (Time Spent in Each Phase of the Cell Cycle)o Purpose 1-2 sentences describing the overall goal of PART B of the experiment; use

complete sentenceso Lab Procedure Write a procedure for PART B. Use your own words and put in numbered

steps. o Data Tables Either recopy or cut out and paste Tables 1.3 and 1.4 into your lab notebook

Day 2:

Before class: Complete Part A of the lab and do the PreLab for Part B (see above)

In class: Get a card of an onion root tip and do Part B of the lab Make sure to do your calculations so you can complete Table 1.4 Finish your Analysis Questions for Part B

Day 3:

Before class: Do the PreLab for Part C

o Subtitle Part C (Effect of Environment on Mitosis)o Purpose 1-2 sentences describing the overall goal of PART C of the experiment; use

complete sentenceso Lab Procedure Write a procedure for PART C. I know there is no actual procedure written

out in the lab for this part, but just read over Part C and write a few sentences about what you will be doing for this section of the lab

o Data Tables Either recopy or cut out and paste Tables 1.5, Table 1.6, and Table 1.7 into your lab notebook

In class: Finish Part B with the Mitosis Cards Work on Part C and do the Analysis Questions

Day 4:

Before class: Do the PreLab for Part D (Crossing Over in Sordaria)

o Subtitle Part D (Crossing Over in Sordaria)o Purpose 1-2 sentences describing the overall goal of PART D of the experiment; use

complete sentenceso Lab Procedure Write a procedure for PART D. Use your own words and put in numbered

steps. o Data Tables Either recopy or cut out and paste Table 1.10 into your lab notebook

In class: Finish Part C and begin Part D of the lab

Day 5: In class:

Finish Part D of the lab and do the analysis Begin PreLab for Part E (HeLa Cells)

o Subtitle Part E (HeLa Cells)o Purpose 1-2 sentences describing the overall goal of PART E of the experiment; use

complete sentenceso Lab Procedure Write a procedure for PART E. Use your own words and put in numbered

steps. o Analysis Questions Rewrite Questions #1-3 in your lab notebook and then answer them;

when Part E is assigned, complete Analysis Questions #4-10

Mitosis and Meiosis Lab

INTRODUCTION

One of the characteristics of living things is the ability to replicate and pass on genetic information to the next generation. Cell division in individual bacteria and archaea usually occurs by binary fission. Mitochondria and chloroplasts also replicate by binary fission, which is evidence of the evolutionary relationship between those organelles and prokaryotes.

Cell division in eukaryotes is more much complex. It requires the cell to manage a complicated process of duplicating the nucleus, other organelles, and multiple chromosomes. This process, called the cell cycle, is divided into three parts: interphase, mitosis, and cytokinesis. This is a picture of the cell cycle:

Interphase is separated into three functionally distinct stages. In the first growth phase (G1), the cell grows and prepares to duplicate its DNA. In synthesis (S), the chromosomes are replicated; this stage is between G1 and the second growth phase (G2). In G2, the cell prepares to divide. In mitosis, the duplicated chromosomes are separated into two nuclei. In most cases, mitosis is followed by cytokinesis, when the cytoplasm divides and organelles separate into daughter cells. This type of cell division is asexual and important for growth, renewal, and repair of multicellular organisms. You end up with two cells that are genetically identical to the parent cell.

Cell division is tightly controlled by complexes made up of several specific proteins. These complexes contain enzymes called cyclin-dependent kinases (CDKs), which turn on or off the various processes that take place in cell division. CDK partners with a family of proteins called cyclins. One such complex is mitosis-promoting factor (MPF), sometimes called maturation-promoting factor, which contains cyclin A or B and a cyclin-dependent kinase (CDK). CDK is activated when it is bound to cyclin, interacting with various other proteins that, in this case, allow the cell to proceed from G2 into mitosis. The levels of cyclin change during the cell cycle. Cyclin levels rise and fall throughout the cell cycle so they are only present at high concentrations when the cell is ready to advance to the next phase of the cell cycle. After the cell has advanced, cyclins are rapidly degraded until they are needed again. So, CDKs can only be active when the levels of cyclin are high enough. If the cell goes all the way through mitosis, usually cytokinesis follows.

Different CDKs are produced during each of the phases of mitosis. The cyclins determine which processes in cell division are turned on or off and in what order by CDKs. As each cyclin is turned on or off, CDK causes the cell to move through the stages in the cell cycle.

Cyclins and CDKs do not allow the cell to progress through its cycle automatically. There are three checkpoints a cell must pass through: the G1 checkpoint, G2 checkpoint, and the M checkpoint. At each of these checkpoints, the cell checks that it has completed all of the tasks needed and is ready to proceed to the next step in its cycle. Cells pass the G1 checkpoint when they are stimulated by appropriate external growth factors; for example, platelet-derived growth factor (PDGF) stimulates cells near a wound to divide so that they can repair the injury. The G2 checkpoint checks for damage after DNA is replicated, and if there is damage, it prevents the cell from going into mitosis. The M (metaphase) checkpoint assures that the mitotic spindles (or microtubules) are properly attached to the kinetochores (anchor sites on the chromosomes). If the spindles are not anchored properly, the cell does not continue on through mitosis. The cell cycle is regulated very precisely. Mutations in cell cycle genes that interfere with proper cell cycle are found very often in cancer cells.

OBJECTIVES

• To describe the events in the cell cycle and how these events are controlled• To explain how DNA is transmitted to the next generation via mitosis• To explain how DNA is transmitted to the next generation via meiosis followed by fertilization• To understand how meiosis and crossing over leads to increased genetic diversity, which is necessary

for evolution

PRELAB QUESTIONS 1. Name 4 reasons why cells undergo mitosis. 2. Why do cells undergo meiosis? 3. Using a prepared slide of an onion root, where would you expect to see the most cells undergoing

division? 4. If you count 90 cells and 30 of them are in prophase, what is the percentage of cells in prophase? 5. If that particular cell takes 60 minutes to undergo mitosis then how much time do you expect that it

would spend in prophase? 6. What are the differences between cell division in plant and animal cells?7. What is Sordaria fimicola? 8. Tell if each combination of spores described below is indicative of a crossed over or non-crossed over

state: a. 8 spores in a 2 tan : 2 wild type : 2 tan : 2 wild type pattern b. 8 spores in a 4 tan : 4 wild type pattern c. 8 spores in a 2 tan : 4 wild type : 2 tan pattern

9. If genes cross over 20% of the time, then how many map units must separate the two genes?10. Who is Henrietta Lacks and why is she significant?

---------------------------------------------------------------------------------------------------------------------------------------PART A Online Mitosis Practice with Onion Root Tip and Mitosis Drawings

All new cells come from previously existing cells. New cells are formed by the process of cell division, which involves both division of the cell’s nucleus (karyokinesis) and division of the cytoplasm (cytokinesis).

There are two types of nuclear division: mitosis and meiosis. Mitosis typically results in new somatic (body) cells. Formation of an adult organism from a fertilized egg, asexual reproduction, regeneration, and maintenance or repair of body parts are accomplished through mitotic cell division (mitosis). You will study mitosis in the first part of this lab.

Where does one find cells undergoing mitosis? Plants and animals differ in this respect. In higher plants the process of forming new cells is restricted to special growth regions called meristems. These regions usually occur at the tips of stems or roots. In animals, cell division occurs anywhere new cells are formed or as new cells replace old ones. However, some tissues in both plants and animals rarely divide once the organism is mature.

To study the stages of mitosis, you need to look for tissues where there are many cells in the process of mitosis. This restricts your search to the tips of growing plants, such as the onion root tip, or, in the case of animals, to developing embryos, such as the whitefish blastula.

In this part of the lab, you will examine the root tip of an onion. The tips of roots are consistently growing, so looking at the cells in the root tip offers a great opportunity to see cells in every stage of mitosis. Roots consist of different regions (see Figure below). The root cap functions in protection. The apical meristem is the region that contains the highest percentage of cells undergoing mitosis. The region of elongation is the area in which growth occurs. The region of maturation is where root hairs develop and where cells differentiate to become xylem, phloem and other tissues.

PROCEDURE PART A: Online Activity- Onion Root Tip Mitosis

1. Go to this website: Onion Root Tip Activity http://www.biology.arizona.edu/cell_bio/activities/cell_cycle/cell_cycle.html

2. On the second page of this website (you can get there by clicking “Next”) it shows you a picture of an onion cell in each phase of mitosis. It also offers an explanation as to what is happening in each stage. Look over this page, and then complete Table 1.1 of your lab.

3. After completing Table 1.1, you can continue with the web activity where you will identify how many cells are in which phase of mitosis. Once you have identified all 36 cells, put the totals in Table 1.2 and calculate the % spent in each phase. The way to do this is to divide the number of cells in each phase by the total number of cells (in this case, 36).

Table 1.1: Plant Cells in Phases of MitosisMitotic Phase Description of What

is happening (in words)

Schematic Drawing w/4 chromosomes

(this is not shown on the website- use your

notes or textbook)

Drawing from Web Activity of the Onion

Root Tip

Interphase

Prophase

Metaphase

Anaphase

Telophase

Table 1.2: Length of Time Spent in Each Cycle

Interphase Prophase Metaphase Anaphase Telophase TOTALNumber of Cells

% of Cells

ANALYSIS QUESTIONS PART A: Online Activity- Onion Root Tip Mitosis1. What phase of the cell cycle do cells spend most of their time in? 2. What phase of mitosis do cells spend most of their time in? 3. If a cell has 24 chromosomes in prophase, how many chromosomes does it technically have in

anaphase? 4. If a cell has 24 chromosomes in prophase, how many chromosomes does each new cell have after

cytokinesis? 5. What is the role of the centrosome? 6. If you found 5 cells out of 100 cells undergoing metaphase, then what is the percentage of cells in

metaphase? 7. If that cell type usually takes 120 minutes to undergo mitosis, then how long do you think it must take to

undergo metaphase? ---------------------------------------------------------------------------------------------------------------------------------------PART B Time Spent in Each Phase of the Cell Cycle

In this part of the lab, you will look at a card that has pictures of cells of an onion root tip (the meristematic region). This card is what you would see if you looked through a microscope, but it is larger, and therefore easier to analyze. Each card represents a “field of view” that you would see in a microscope. Based on your practice from Part A, you will be able to identify what phase of mitosis each cell is in. Each person in your lab group will do a card individually, and then you will pool your results to do your calculations. This will help you estimate the relative length of time that a cell spends in the various stage of cell division. The length of the cell cycle is approximately 24 hours (1,440 minutes) for cells in actively dividing onion root tips.

PROCEDURE PART B: Time Spent in Each Phase of the Cell Cycle

1. Obtain a card with a field of view from a dividing onion root tip. On your individual card, look at each cell and determine what phase of mitosis it is in, and put a tally mark for each cell in Table 1.3.

Table 1.3: Individual Field of View for Onion Root Mitosis – Cell Counts

Tally Marks for Each CellTotal # of

Cells Spent in Each Phase

Interphase

Prophase

Metaphase

Anaphase

Telophase

2. Record your TOTALS from Table 1.3 in Table 1.4 under Field of View #1. Share your information with 3 other lab group members. Obtain their data and fill that in under Field of View #2 through #4 and add it all up to get an overall TOTAL. Record that data in Table 1.4 as well.

Table 1.4: Totals for Cells Spent in Each Phase of Mitosis

Number of Cells % of Total Cells

Counted

Time in Each Stage

Field of View #1

Field of View #2

Field of View #3

Field of View #4

TOTAL

InterphaseProphaseMetaphaseAnaphaseTelophase

Total Cells Counted = ______

3. Calculate the % of total cells counted by dividing your totals for each phase by the total number of

cells counted. Record this information in Table 1.4. 4. Calculate the Time Spent in Each Stage using the following equation and complete Table 1.4:

% of cells in each Stage x 1, 440 minutes = ____ minutes of the cell cycle spent in that stage

ANALYSIS QUESTIONS PART B: Time Spent in Each Phase of the Cell Cycle

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 Table 1.4, what can you infer about the relative length of time an onion spends in each stage of cell division?

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PART C Effects of Environment on Mitosis

Scientists reported that a fungal pathogen may negatively affect the growth of soybeans (Glycine max). Soybean growth decreased during three years of high rainfall, and it was discovered that the soybean roots were poorly developed in those years. Close relatives of the R. anaerobis are plant pathogens and grow in the soil. A lectin-like protein was found in the soil around the soybean roots. This protein may have been secreted by the fungus. Lectins induce mitosis in some root apical meristem tissues. In many instances, rapid cell divisions weaken plant tissues.

In Part C of this experiment, we are going to do a dry lab. That means that I will provide you with data and you will do the analysis (instead of actually doing a lab with both treated and untreated lectin-exposed roots). We are going to try to figure out if the roots that are treated with lectin contain cells that are more likely to be in mitosis or if the protein has no effect. The roots that have been treated have soaked in lectin for 18 hours. Cells from both the treated and untreated roots have been put on a slide and stained so that we can see what stage the cell is in. Each of the cells have been identified as in either Interphase or in Mitosis. The data is below.

Table 1.5 has the observed values of the number of cells in interphase vs. the number of cells in mitosis for both untreated roots (the control) and the lectin treated roots. Add up the numbers and fill in the Totals for each category.

Table 1.5: Table of OBSERVED Values of Cells in Interphase and Mitosis for Treated and Untreated Roots

Interphase Mitosis TOTALControl (Untreated) 148 25

Lectin Treated 161 88TOTAL

The expected values have been calculated for you in Table 1.6.

Table 1.6: Table of EXPECTED Values of Cells in Interphase and Mitosis for Treated and Untreated Roots

Interphase MitosisControl (Untreated) 127 46

Lectin Treated 182 67

Recall that the equation for chi square is

X2 = (Observed – Expected) 2 Expected

Fill in Table 1.7 to calculate the Chi Square for this data . First, copy the observed and expected values from the tables above. Next, do the calculations. Once you figure out the chi square, you can determine whether the null hypothesis is accepted or rejected.

Table 1.7: Calculation of Chi-Square Values

Observed (o)

Expected (e)

(o-e) (o-e)2 (o-e)2/e

Control InterphaseControl MitosisLectin Treated InterphaseLectin Treated Mitosis

Chi Square =

Look at the Critical Value of the Chi Square Distribution Table:

Critical Values of Chi Square DistributionProbability Degrees of Freedom (df)

1 2 3 4 5

0.05 3.84 5.99 7.82 9.49 11.10.01 6.64 9.21 11.3 13.2 15.10.001 10.8 13.8 16.3 18.5 20.5

The degrees of freedom (df) equals the number of groups minus 1. In this case, there are four groups so the degrees of freedom is 3. The p value is 0.05, and the critical value is 7.82. If the calculated chi square value is greater than this critical value, then the null hypothesis is rejected. If the calculated chi square value is less than this critical value, the null hypothesis is accepted (“not rejected”).

ANALYSIS QUESTIONS PART C: Effects of Environment on Mitosis

1. What is lectin and how does it end up in the soil? 2. What effect does lectin have on plant tissues? 3. What is the null hypothesis for Part C of this lab? 4. What is the equation for chi square? 5. What did you get for your chi square value? 6. Did you accept or reject the null hypothesis and why? 7. Does lectin have a significant effect on the rate of mitosis in roots?

---------------------------------------------------------------------------------------------------------------------------------------PART D Crossing Over in Sordaria

Sordaria fimicola is a fungus that can be used to demonstrate the results of crossing over during meiosis. Sordaria is a haploid (n) organism for most of its life cycle. It becomes diploid (2n) only when the fusion of the mycelia (filament like groups of cells) of two different strains causes 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 one mitotic division, in Sordaria results in the formation of eight haploid ascospores contained within a sac called an ascus (plural, asci). See the life cycle of Sordaria fimicola below:

Recall that crossing over is when segments of two chromatids from homologous chromosomes exchange genetic information. This leads to recombinant offspring. To observe crossing over in Sordaria, one must make hybrids between wild type (normal) and mutant strains of Sordaria. Wild type Sordaria have black ascospores (+). The mutant strain that we will be working with 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 1.8, no crossing over has occurred. In Figure 1.9, it shows the results of crossing over between the centromere of the chromosome and the gene for ascospore color.

Figure 1.8. No Crossing Over in Sordaria

Figure 1.9. Crossing Over in Sordaria

As the two pictures show, if you have:

4 tan then 4 black NO CROSSING OVER

2 tan, 2 black, 2 tan, 2 black2 black, 2 tan, 2 black, 2 tan CROSSING OVER OCCURRED2 tan, 4 black, 2 tan (these are also called RECOMBINANT)2 black, 4 tan, 2 black

Figure 1.10. Crossing over vs. Non-Crossing Over in Sordaria

In this part of the lab, you will be examining a prepared slide of Sordaria. Two strains, wild type and tan, have been put together on agar and allowed to combine. What you are seeing on the slide is the result. You will count how many of the hybrid (those that have both tan and black) asci have crossed over, and how many have not. From that data, you will calculate the frequency of crossing over. The frequency of crossing over appears to be determined largely by the distance between genes, in this case, between the gene for spore coat color and the centromere. The probability increases as the distance between those genes becomes larger. The frequency of crossover, therefore, appears to be directly proportional to the distance between the 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.

PROCEDURE Part D: Crossing Over in Sordaria

1. For each group, get two microscopes and two slides of Sordaria. 2. Get with a partner, and one person can call out crossed over or non-crossed over, and the other partner

can keep track. It is hard to do it yourself because once you look away from the microscope, it is difficult to find where you left off. So, keep your eye on the slide, and have someone else record the data. Have two other group members do this for the other Sordaria slide. This is what your slide should look like.

3. Once you tally how many crossed over and non-crossed over you have, have each set of partners fill in Table 1.10 with your data.

4. Using the data from Table 1.10, determine the distance between the gene for spore color and the centromere. Calculate the percentage of crossovers by dividing the number of crossover asci (2:2:2:2 or 2:4:2) by the TOTAL number of asci and multiply by 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 (each one goes through mitosis, so you get 2 for each original one). Record your results in Table 1.10.

Table 1.10. Crossing over in Sordaria.

Number of Non-

Crossing Over (4:4)

Number of

Crossing Over

(2:4:2 or 2:2:2:2)

Total Asci

% of Asci

showing Crossing

Over

Map Units from gene

to centromere(% of Asci

showing Crossing

over DIVIDED

BY 2)Slide #1Slide #2TOTAL

ANALYSIS QUESTIONS PART D: Crossing Over in Sordaria

1. What is crossing over? What process does this occur in? 2. How do you figure out how far apart a gene is from the centromere?

3. How can you tell if a Sordaria spore has crossed over or not? What would it look like if it had? If it hadn’t?

4. What is another term for Sordaria that has been crossed over? 5. The published map distance between the spore color gene and the centromere is 26 map units. What

was your answer for map units? How does that compare to the published number? ---------------------------------------------------------------------------------------------------------------------------------------

Part E HeLa Cells

Cancer cells go through uncontrollable cell division. They do not stop when normal cells would stop and they do not show density dependent inhibition. Cancer can be caused by several things. Some of those causes are genetic factors, lifestyle factors (tobacco, diet, physical activity), viruses (HPV can cause cervical cancer), and environmental exposures to different types of chemicals and radiation.

Some of the most famous cancer cells that are used in research today, and have been used over the past 60 years, are HeLa cells. These are cells from a woman named Henrietta Lacks who had cervical cancer. Doctors removed her tumor and took cells without her consent, and these cells have been called immortal because they are still dividing in labs today.

PROCEDURE PART E: HeLa Cells

1. Copy and answer the Analysis Questions #1-3 in your lab notebook. 2. Read the article on Henrietta Lacks and her HeLa cells in the link below. 3. Copy and answer the Analysis Questions #4-10 in your lab notebook.

Article on Henrietta Lacks: http://www.smithsonianmag.com/science-nature/henrietta-lacks-immortal-cells-6421299/?no-ist (this article is also available on the wiki in word format if the online version is not working)

ANALYSIS QUESTIONS PART E: HeLa Cells

Answer the following questions before reading the article: 1. What is cancer? 2. What are some causes of cancer? 3. What is density dependent inhibition?

Answer the following questions while reading the article: 4. Who is Henrietta Lacks? 5. Why are HeLa cells so important and what are some of the discoveries they have been partially

responsible for? 6. Where did they come up with the name “HeLa” cells? 7. Where was Henrietta raised? 8. How did HeLa cells contaminate other cell cultures? 9. How did Henrietta’s husband understand what the scientists were saying when they reached out to get

DNA samples from him? 10. Why did Henrietta’s sons get interested in HeLa cells?