ap biology lab-04

1Copyright 2013 Quality Science Labs, LLC

LAB 4Cell Division: Mitosis and Meiosis

Big Idea 3: Genetics and Information Transfer

How do eukaryotic cells divide to produce genetically identical cells to produce gametes with half the normal DNA?

Please be sure you have read thestudent intro packet before you do this lab.

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations SummaryPre-lab and Questions: Loss of Cell Cycle Control in CancerLab Investigation 4.1: Environment Effects on Mitosis

Part 1 - How does the environment affect mitosis?Null hypothesis and Chi-squared data analysis for significance

Part 2 - Student Guided Inquiry

Lab Investigation 4.2: Meiosis and Crossing Over in Sordaria

2 Copyright 2013 Quality Science Labs, LLC

LAB 4 - Cell Division: Mitosis and Meiosis

Big Idea 3: Genetics and Information TransferHow do eukaryotic cells divide to produce genetically identical cells to produce

gametes with half the normal DNA?

BACKGROUND

What are the differences between mitosis and meiosis?Mitosis and meiosis are two of the most fantastically amazing processes in

life. Mitosis is a process in the cell cycle that allows the cell to duplicate without fertilization. Prokaryotes (like bacteria) basically separate the cell contents into two parts. This is called binary fusion. In eukaryotes, the process is more complex and occurs in two main stages: mitosis and cytokinesis. Mitosis is the division of the cell nucleus which is followed by the division of the cytoplasm or cytokinesis. A common misconception is that interphase is the first stage of mitosis. However, since mitosis is the division of the nucleus, prophase is actually the first stage. Reproduction by mitosis is classified as asexual since the new daughter cells are identical to the parent cell. Mitosis occurs as organisms grow and develop, like

Mitotic Phase

First Grow

th Phase

Synthesis Phase

Seco

nd G

row

th P

hase

Interphase

Telophase

Anaphase

Metaphase

Prophase

Growth and normal metabolic roles

DNA replication

Growth andpreparationfor mitosis

MG1

S

G2

G2 CheckpointM Checkpoint

G1 Checkpoint(Restriction)

The Cell CycleCyclin Checkpoints in red


human skin cells that are being constantly replaced.Meiosis involves two nuclear divisions: Meiosis I is the reduction

division. It reduces the chromosome number from diploid to haploid and separates the homologous pairs. While different, meiosis I looks similar to mitosis. There is crossing over of alleles from the chromatids or recombinants, so the chromosomes are not identical to the parent as in mitosis. Meiosis II, the second division, separates the sister chromatids and results in four haploid gametes. Meiosis is how sexually reproducing organisms produce gametes.

Cell Cycle, Cell Division, and MitosisThe cell cycle is a series of events that cells go through as they

grow and divide. During the cell cycle, a cell grows, prepares for division, and divides to form two daughter cells identical to the mother cell. The purpose of mitosis is tissue growth, regeneration, or asexual (vegetative) reproduction. There are three main stages during the cell cycle: interphase, karyokinesis (mitosis), and cytokinesis.

Interphase is the longest lasting and includes three phases. G1 phase is a period of activity in which cells do most of their growing. Cells increase in size and synthesize new proteins and organelles. S phase is when DNA is replicated and key proteins associated with the chromosomes are synthesized. During the G2 phase, many of the organelles and molecules required for cell division are produced. The M phase or mitosis (karyokinesis) now begins the process of cell division.

Mitosis is divided into four main phases: prophase, prometaphase and metaphase, anaphase, and telophase. Finally, cytokinesis occurs which divides the cytoplasm and results in the production of the two identical daughter cells to the parent cell.

How is the Cell Cycle Regulated?In the early 1970's, a series of experiments confirmed the cell cycle

is controlled by specific signaling molecules in the cytoplasm that triggers and coordinates key events. In the cells cycle control system, there are three main checkpoints where stop and go-ahead signals regulate the cycle. These signals are both internal and external. The G1 checkpoint is the most important. If the cell receives the go-ahead signal, it will usually complete the G1, S G2,and M phases and divide(exception include mature muscle and nerve cells which never divide, but are in the G0 phase.

Cell division is strictly controlled by specific protein complexes called cyclins containing enzymes called cyclin-dependent kinases (CDKs). CDKs can turn on or off the different processes involved in cell division. There are CDKs specific for each phase of the cell cycle and their levels change during the cycle. CDK is activated when it is bound to cyclin. As each cyclin is turning on or off, CDK causes the cell to move through the stages in the cell cycle.

Looking back at the cell cycle diagram, there are three checkpoints

Prophase

Metaphase

Anaphase

Telophase


that cyclins and CDK control before allowing the cell to proceed to the next phase: G1, G2, and Mitosis at metaphase and anaphase. For example, the G2 checkpoint checks for damage after DNA is replicated, and if there is damage, it prevents the cell from going into mitosis. Cancer cells often are found to have the cell cycle genes that interfere with proper cell cycle control.

Fertilization, Meiosis, and Crossing Over of ChromosomesMeiosis involves the reduction division of chromosomes in phase I (Meiosis I)

resulting in two diploid daughter cells, but pieces of the chromosomes have been exchanged (crossing-over). During this phase, homologous chromosomes form a tetrad. Crossing-over occurs where chromatids cross over on another and the crossed sections of the chromatids are exchanged. The homologous chromosomes

separate and two new cells are formed. Although they look like the result of mitosis, they are different with sets of chromosomes and alleles that are different from each other (and from the original diploid cell) after crossing over occurred. Phase II (Meiosis II) has the chromosomes lining up in the center of each cell and separating. Each of the four daughter cells produced receive two chromatids and are haploid. Each daughter cell is genetically different from one another and from the original cell. These haploid cells are referred to as gametes. In animals, the female gamete is an egg and the male gamete is sperm. After fertilization, then mitosis begins as the cells in the fertilized egg undergo cell divisions. See the figure to the left.

1 2 3 4 5

6

7

8

9

10

11}Meiosis Legend

1. Interphase G 2. Interphase S 3. Prophase I 4. Metaphase I 5. Anaphase I 6. Telophase I

7. Prophase II 8. Metaphase II 9. Anaphase II 10. Telophase II 11. Cytokinesis

Meiosis


PREPARATIONMaterials and equipment are listed with each lab separately.

Timing and length of lab activities Lab Investigation 4.1 needs about five days prep for growth of onion roots.

During the last three days, the onions will be exposed to the variable caffeine. Two lab periods will be required for staining and preparing slides, collecting data, and mathematical analysis. Additional two lab periods are needed for the guided inquiry. The Pre-lab cancer cell analysis and the Lab Investigations 4.2 do not require lab bench time, or if done in class require one class period each.

Learning objectives aligned to standards and science practices (SP)

To describe the events that occur in the cell cycle (3A2 and SP 1.2).To construct an explanation, using visual representations, as to how the DNA

in chromosomes is transmitted to the next generation via mitosis, or meiosis followed by fertilization (3A2 and SP 6.2).

To represent the connection between meiosis and how crossing over leads to increased genetic diversity (3A2 and SP 7.1).

General safety precautionsStudents should wear gloves and safety goggles or glasses when handling the

chemicals and razor blades. To avoid injuries, students should use a pencil eraser instead of their thumbs when pressing down on cover slips.


Pre-lab and Questions: Loss of Cell Cycle Control in Cancer

Perhaps you know someone who has battled cancer in some form. Cancer can occur when cells lose control of their cell cycle and divide abnormally. This happens when tumor-suppressant genes, such as p53 or Rb (retinoblastoma), are mutated. In normal cells, mitosis usually is blocked if there is DNA damage. Sometimes, however, DNA damage makes cells divide more often as with certain forms of leukemia.

Consider the following questions before you start this investigation:How is the cell cycle controlled in normal cells? (See background info at the

beginning of this lab.)What are cyclins and CDKs? What do these proteins (enzymes) do in a cell?How are normal and cancer cells different from each other?What are the main causes of cancer?What goes wrong during the cell cycle in cancer cells?What makes some genes responsible for an increased risk of certain cancers?

Do you think the chromosomes might be different between normal and cancer cells?

Background on HeLa cellsHeLa cells are cervical cancer cells

first isolated from a woman named Henrietta Lacks. Her cells have been cultured since 1951 and used in numerous scientific experiments. Henrietta Lacks died from her cancer not long after her cells were isolated. Lacks cancer cells contain remnants of HPV (human papillomavirus), which we now know increases the risk of cervical cancer.

Materials:Human karyotype pictures above and on the next page of

normal and cervical HeLa cancer cells.

Human karyotype (normal cells) from www.daviddarling.info/encyclopedia/K/karyotype.html


Human karyotype (HeLa cervical cancer cells) from Modal karyotype of the HeLa Ohio cervical adenocarcinoma cell line htcl.cytspb.rssi.ru


Procedures: 1. Write a hypothesis as to how the chromosomes of a cancer cell

might appear in comparison to a normal cell and how those differences are related to the behavior of the cancer cell.

2. Compare the normal karyotype picture to the HeLa cell karyotype. Count the number of chromosomes in each type of cell and note any differences in their appearance.


QuestionsHeLa Cells:

1. Do your observations support your hypothesis?

2. If not, what type of information might you need to know in order to understand your observations?

3. If yes, what type of information can you find that would validate your conclusions?


Lab investigation 4.1: Part 1 - Environmental Effects on Mitosis

How do environmental factors affect the rate and quality of mitotic division? Scientists are interested in this question from the perspective of disease, specifically, the uncontrolled division of cells known as cancer. This investigation will allow you to make a simplified study between the relationship between environment and mitosis. You will test the rate of growth of onion roots when exposed to an environmental chemical, caffeine in the form of coffee.

Hypothesis: Make a hypothesis to describe the effect of caffeine on the stages of mitotic division.

Materials:Onion bulbs (2)* Paper towels*Toothpicks (8) Distilled water*Glass canning jars (2)* Microscope slides (4)

Concentration of caffeine (coffee): 0.5% (0.5 g/100 mL)*

Microscope coverslips (4)Microscope*

Metric rulers (2) 0.5% Toluidine Blue stainDigital scale 0.1 M HCl (hydrochloric acid)Razor blade, straight edge Light bulb heat source*Forceps Stopwatch

*items not included


Procedures:Advance Preparation:

Grow onion bulb roots in water to 1 cm a few days before experiment starts.

1. Afteronionrootshavereached1cm,changeoneofthejarsfromwatertocaffeine;monitoronionrootgrowthinthewaterandcaffeinejarsovera three day period. Record daily measurements in centimeters of the root lengths.

2. After the third day of growth in caffeine and water, harvest and stain the onion root tips. If you wait longer, the cells may not show much mitotic activity.

3. Using the scalpel, cut five 1-2 mm pieces of root tips from the onion in the water and place them on a slide. Add five drops of 0.1 M HCl to the root tips on the slide. Place the slide about 8 cm from a 45 watt light bulb for 30 minutes. When the HCl starts to dry up, add a few drops more. (The base of a lava lamp works great. See photos to the right.)

4. Remove the slide from the heat source. Tear a paper towel into pieces and using the ragged edges, dab and soak up the excess HCl.

5. Next, add 3-5 drops of 0.5% Toluidine Blue stain to the slide with the five root tips. Return the slide to the heat source for five minutes. Again, if the stain starts drying out, add one or two more drops.

6. Remove the slide from the heat source, dab the excess stain and add another two to three drops


of stain. Transfer two root tips from the slide to a new slide, making sure there is a bubble of stain covering the root tips. Add the cover slip (at a 45 degree angle to minimize air bubbles) and proceed to the following squash technique:

7. Place the slide on a paper towel cushion and cover the slide and coverslip with a piece of paper towel. Push down on the coverslip with the eraser of a pencil. You can press the eraser of the pencil rather hard as long as you do not twist or torque. Press straight down over the section that has the root tip.

8. Repeat steps 3 to 7 for your caffeine exposed root tips slide staining. 9. Look at your slides under a microscope at low and high powers

for cells undergoing mitosis. The cells will not be neatly arranged with the squash technique. Examine the size, shape, and position of the chromosomes in each treatment in order to help you identify the different stages of mitosis. Count and record the number of cells in each phase of mitosis on Table 4.1a.

10. Sketch the stages of mitosis observed from roots in each treatment. 11. Analyze the class data with Chi-squared data analysis IF you

have access to the data. Collect the class data for each group and record it in

Table 4.1b. Calculate the mean and standard deviation for each group.

Compare the number of cells from each group in interphase and in mitosis using Table 4.1c.

Use a chi (pronounced ki) squared distribution test to statistically analyze the data in Table 4.1d.

12. Enter the number of caffeine cells in interphase and mitosis as observed (o) in Table 4.1d. Calculate the percentage of cells in interphase and

mitosis in the control group from Table 4.1b. Multiply the percentages by the total number of cells in

the caffeine group; this will give the expected numbers (e). Calculate the chi-square (X2) value for the test. Compare this value to the critical value in Table 4.1e.

13. Enter the number of caffeine cells in interphase and mitosis as observed (o). Calculate the percentage of cells in interphase and

mitosis in the control group from Table 4.1b. Multiply the percentages by the total number of cells in

the caffeine group; this will give the expected numbers (e) in Table 4.1d.

Calculate the chi-square (X2) value for the test (Table 4.1d). Compare this value to the critical value in Table 4.1e.


14. See Table 4.1e. The degrees of freedom (df ) equals the number of groups minus one. In this case, there are two groups, interphase and mitotic; therefore, df = 2-1 or 1. At a p value of 0.05, the critical value is 3.84. If the

calculated chi-square value is greater than or equal to this critical value, then the null hypothesis is rejected and caffeine DOES have a significant effect on the mitotic growth of onion root tip cells.

If the calculated chi-square value is less than this critical value, the null hypothesis is not rejected and caffeine does NOT have an effect on the mitotic growth of onion root tip cells. The NULL HYPOTHESIS says that there is NO EFFECT OF CAFFEINE ON THE MITOTIC GROWTH OF ONION ROOT TIP CELLS.

Conclusions and Post-lab Review: 1. Was there an increase in the number of cells in mitosis in the

caffeine treated roots? If you were able to analyze a large set of data, was the difference significant (at p=0.05)?

2. Why is it important to collect class data when performing chi-square analysis?

3. Does an increase in the number of cells in mitosis mean that these cells are dividing faster than the cells in the roots with the lower number of cells in mitosis?


Number of Mitotic Phases in Each TreatmentInterphase Prophase Metaphase Anaphase Telophase Cytokinesis

Control H2OCaffeine (0.5%)

Table 4.1a: The Number of Cells in Interphase, Mitosis, and Cytokinesis

GroupNumber of Cells

Interphase Mitotic TotalControlCaffeine

Table 4.1b: Class Data

Interphase Cells Mitotic Cells Total

ControlCaffeine

Table 4.1c: Comparison of Interphase and Mitotic Cells

# Observed (o)

# Expected (e) (o-e) (o-e)

2 (o-e)2 / eInterphase CellsMitotic Cells

Table 4.1d: Calculation of Chi-Square X2 = (o-e)2/e =

ProbabilityDegrees of Freedom (df)

1 2 3 4 50.05 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

Table 4.1e: Critical Values of the Chi-Squared Distribution


Part 2 - Student Guided Inquiry, Environmental Effects on Mitosis

Going back to Lab Investigation 4.1 on the environmental effects on mitosis and plants, what other factors might increase or decrease the rate of mitosis? Design and conduct an investigation to determine the effects of such factors as: salinity, temperature, pH (acid rain), humidity, fertilizers: N, K, P, Fe, sugar, CO2; or biotic organisms like beetles, ants, roundworms, etc. which might alter root growth.

Step 1: Chose your driving question to investigate.Step 2: Fill in the ExD (Experimental Design) form on the next page

to plan your experiment. Remember to include a control and to test only one variable at a time while keeping all other potential variables constant.

Step 3: Design data tables and collect data.Step 4: Analyze the data (graphs chi-square calculations).Step 5: Summarize your conclusions.


Experimental Design (ExD) FormComplete this ExD pre-lab planning form before beginning your lab

1. Independent variable: (What is the cause agent? What are you changing?)

2. Dependent variable: (What is being measured?)

3. Lab set-up:

ExperimentalGroups

Number of Trials

4. Control: (What is the experimental group being compared to?)

5. Hypothesis: (Use an if [Independent Variable], then [Dependent Variable] format. State the cause and eect relationship between the I.V. and the D.V. It must be testable.)

6. Lab title: (e eect of ____[I.V.] ____on ____[D.V.]____.)

7. Experimental constants: (Name at least six variables NOT altered during the experiment.)

8. Sketch of experimental set up with labels:

9. Detailed procedure:


LAB Investigation 4.2: Meiosis and Crossing-Over in Sordaria

The fungus Sordaria fimicola is an ascomycete fungus that can be used to demonstrate the results of crossing over during stage 1 of meiosis. Sordaria is a haploid organism for most of its life cycle. It becomes diploid only when the fusion of the mycelia (filament-like groups of cells) of two different strains results in the fusion of the two difference 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 (pleural, asci). A single gene determines the spore color. In the illustration on the following page (Figure 4.2d), a cross was made between wild type (black) and tan strains. The resulting zygote produces either parental type asci, which have four black and four tan spores in a row; or recombinant asci, which do not have this pattern (refer to Figures 4.2b and 4.2c).

Mature peritheciumcontaing many asci

8 haploid nuclei

4 haploid nuclei

MitosisMeiosisDiploid zygote

FertilizationMycelia fuse

Mycelium

Mitosis Filament

Mitosis

Ascospore

Spore discharge

Ascospores

Meiosis without crossing overFigure 4.2b

Meiosis with crossing overFigure 4.2c

The Life Cycle of Sordaria fimicolaFigure 4.2a

or

or or or


Sordaria fimicola asci tetrad analysisFigure 4.2d

Materials:Figure 4.2d above, an illustration of Sordaria asci

Procedures: 1. For at least 50 asci in Figure 4.2d above, count the number

showing crossover and the number not showing crossover. Enter your data in Table 4.2a.


Number of 4:4 (non-crossovers)

Number of asci showing crossover

Total Asci

% Asci Showing Crossover Divided by 2

Gene to Centromere Distance(map units)

Table 4.2a

Data Analysis:The frequency of crossing over appears to be governed by the distance between

the genes, or in this case, between the gene for spore coat color and the centromere. The probability of a crossover occurring between two 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 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 can use the map unit.

Using your data from Table 4.2a above, determine the distance between the gene for spore color and the centromere:

1. Calculate the percentage of crossovers by dividing the number of crossover asci by the total number of asci X 100.

2. To calculate the map distance, divide the percentage of crossover asci by 2. Record your results in Table 4.2a.


Evaluation and Conclusions:

1. Why did you divide the percentage of asci showing crossover (recombinant) by 2?

2. The published map distance between the spore color gene and the centromere is 26 map units. How did your data (or class data) compare with this distance?

3. Illustrate what happened during meiosis to produce the results you found.

ap biology lab-04

Documents

cell division

parent cell

cell nucleus

mother cell

cell contents

stage of mitosis

purpose of mitosis

mitosisthe cell cycle