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LAB TOPIC 9     Chromosomes and Cell Division From Dickey, Jean Laboratory Investigations for Biology (2nd edition) (2003, Benjamin Cummings

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LAB TOPIC 9

 

 

Chromosomes and Cell Division

From Dickey, Jean Laboratory Investigations for Biology (2^nd edition) (2003, Benjamin Cummings.)

 

 

Before you begin the activities in this lab topic, you should be able to

 

1. Explain the role of DNA in genetics.
2. Explain how the eukaryotic genome is organized. 
3. Define the terms chromosome and gamete.
4. Distinguish the general purpose of mitosis from the purpose of meiosis.

 

Introduction

The genetic information of all organisms is found in deoxyribonucleic acid (DNA), which consists of varying sequences of the four nucleotides adenine, thymine, guanine, and cytosine. Discrete sections of DNA are called genes, and they are the "blueprints" that make organisms. In the cells of eukaryotes such as wheat and humans, DNA is packaged with proteins. Most of the time this DNA-protein complex is found in a threadlike form called chromatin. Recall Lab Topic 5 when you looked at your cheek cells under the microscope. The most prominent feature of the cells was the nucleus, but you needed to use the high-power objective to see it clearly. If the DNA molecules in one of those microscopic nuclei were unwound, attached together, and pulled out like a string, it would be two meters long! It fits inside the nuclei because it is extremely thin. In this lab topic, you will extract DNA from wheat cells to see this famous molecule for yourself.

 

During cell division, the chromatin is elaborately wound up into the coiled structures called chromosomes. When cells give rise to new cells, there must be a way for each new cell to receive an exact copy of all of the chromosomes. In this lab topic you will examine how the process of mitosis accomplishes this.

 

Genetic information must also be passed from parents to offspring, and in this lab topic you will see how the process of meiosis distributes chromosomes into gametes for the purpose of sexual reproduction.

Outline

Exercise 9.1:  The Genetic Material

          Activity A:  Extracting DNA from Cells

           Activity B:  Karyotypes

Exercise 9.2: The Process of Mitosis

           Activity A: Pop Bead Simulation of Mitosis

           Activity B: Viewing Mitosis in Onion Root Tip Cells

              Activity C: Determine Mitotic Sequence Using Cell Models

Exercise 9.3: The Process of Meiosis

           Activity A: Pop Bead Simulation of Meiosis

           Activity B: Viewing Meiosis in Organisms

 

 

EXERCISE 9.1

The Genetic Material Objectives

After completing this exercise, you should be able to  1.  Describe how DNA can be extracted from cells.

2. Explain what a karyotype shows.
3. Define the following terms: homologous chromosomes, centromere, diploid, and haploid.

 

Activity A: Extracting DNA from Cells

 

DNA is found in almost every cell of every organism. You've heard about it, you've seen diagrams of its structure, but have you ever actually seen DNA molecules? In this activity you will extract DNA from the embryonic cells of wheat seeds, also known as wheat germ. After softening the cell walls of the wheat germ in warm water, detergent is used to break up the membranes. The DNA is then separated from the rest of the cell contents.

           

Procedure

1. Weigh out 1 g of wheat germ.
2. Put the wheat germ in a large (50-ml) test tube.
3. Dip warm water from the container in the water bath and measure 20 ml in a        graduated cylinder. Pour the water into the test tube with the wheat germ.
4. Using a wooden stick, stir the wheat germ gently for 3 minutes.
5. Get a Pasteur pipetful of detergent and release it into the test tube with the wheat         germ.
6. Stir the wheat germ mixture gently for 5 minutes, being careful not to create any        foam.    * Stir slowly to avoid making bubbles!
7. Measure 15 mL of cold alcohol in a graduated cylinder.
8. Tilt the test tube containing wheat germ at a 45-degree angle and very slowly pour the alcohol from the graduated cylinder down the side of the wheat germ tube. The alcohol should just trickle down the side and come to rest on the top of the water so that it forms a separate layer.

* Pour very slowly, taking care not to let the layers mix. 9.  Once you have poured in all the alcohol, place the test tube in its rack and do not move it for at least 15 minutes. The DNA will begin precipitating out immediately between the two layers of liquid. Do Activity B while you wait. 

10.  After 15 minutes, the DNA should be floating on the top of the lower layer, in the layer of alchohol.  Use a wooden stick or hooked pipette to spool it like cotton candy

 

Describe what the DNA looks like.

 

 

The precipitated DNA in the alcohol layer is only part of the total DNA from the wheat cells. Much of the DNA is still in the water below. You can bring this DNA into the alcohol, where it will precipitate. Tilt the test tube slightly and insert the wooden stick into the yellowish water fraction, then pull the stick up into the alcohol. You should see more DNA come up with it. Be careful not to stir the layers together, though! When it reaches the alcohol, the DNA will precipitate in its stringy form and you can spool it, too.

 

Most protocols for DNA extraction use an enzyme to digest proteins. That step was omitted in this procedure to get faster results. How would your results be different if you used a protein-digesting enzyme? (Hint: What protein is DNA tightly wound with in chromosomes? What would DNA be like if it were missing?)

 

Activity B: Karyotypes

 

Although most of the DNA in a prokaryotic organism such as bacteria is carried on a single chromosome, in eukaryotes (animals, plants, fungi, and protists) the DNA is divided among numerous chromosomes. Each eukaryotic species has a characteristic number of chromosomes, but that number does not indicate the complexity of the organism. For example, redwood trees have 66 chromosomes, gypsy moths have 62, yeast have 32, and catfish have 58. We humans have 46 chromosomes in each body cell. Guppies also have 46 chromosomes, but there are many differences in the genetic "blueprints" that make a guppy and the genetic "blueprints" that make a human.

We can study an organism's chromosomes by making a karyotype of them. To make a karyotype, the nucleus of the cell is ruptured to isolate the chromosomes and then a microscopic digital image is made. A technician uses a computer program to arrange the images of the chromosomes in homologous pairs (Figure 9. 1). The pairs are matched by comparing physical characteristics such as length and staining pattern. Homologous chromosomes have these physical similarities because they are genetically similar: they each carry the same genes in the same order.

 

Figure 9.1 shows a finished karyotype of a human male. In humans there are 22 homologous pairs of autosomes (chromosomes that are not concerned with sex determination) plus a pair of sex chromosomes. Females have a homologous pair of X chromosomes, but the male's X and Y, shown here, don't match.

 

 

 

 

 

Figure 9. 1.  Karyotype of human male.

 

The karyotype, as well as other pictures of chromosomes that you have seen in books, shows chromosomes from cells that are undergoing mitosis. Thus the chromosomes are distinct, thickly coiled structures rather than the thready chromatin form. You may also be able to see that the chromosomes in Figure 9.1 are doubled, which makes each one look like a slender "X." Before a new nucleus can be made, the DNA in each chromosome must replicate (make an exact copy) so that each new cell will have exactly the same genetic material as the original cell. After replication, the chromosomes remain attached at a place called the centromere. The point of attachment is not always in the center of the chromosomes. The position of the centromere is another characteristic that technicians use to match homologous pairs.

 

 

How many homologous pairs of chromosomes do the following organisms have?

redwood tree-

 

gypsy moth-

 

catfish-

 

guppy-

 

A cell that contains the correct number of homologous pairs for its species is said to be diploid. A cell with only one member of each homologous pair is said to be haploid. Almost all of the cells in our bodies are diploid. What cells are haploid, and why?

 

 

 

Suppose the karyotype shown in Figure 9.1 was taken from a cheek cell. What cell division process would produce a new cheek cell?

 

 

 

Describe what the karyotype of a new cheek cell from this same individual would look like. That is, how many chromosomes would it have? Would it be haploid or diploid?

 

 

Suppose we made a karyotype of a gamete from this individual. What would it look like? How many chromosomes would it have? Would it be haploid or diploid?

 

 

EXERCISE 9.2

The Process of Mitosis

 

Objectives

After completing this exercise, you should be able to

1. Explain the purpose and location of mitosis in organisms.
2. Describe and recognize the phases of mitosis.

The union of a haploid egg with a haploid sperm produces a diploid zygote. The zygote has a nucleus that holds the chromosomes, which in turn contain the entire blueprint to make the organism. From that single cell, the process of mitosis produces the many (sometimes trillions of) diploid cells that make up the body of a multicellular organism. Therefore each of the organism's cells contains an exact copy of the same genetic material.

 

Because cells must constantly be replaced, mitosis also produces new diploid cells throughout the life of an organism. For example, your entire epidermis is replaced every 15 to 30 days, and your body makes approximately 200 billion new red blood cells every day. That's a lot of mitosis!

 

In the following activities you will examine the steps in mitosis by using models of chromosomes. You will then identify these steps in onion cells.

 

Activity A: Pop Bead Simulation of Mitosis

You will use pop beads to represent chromosomes and follow them through the processes of cell division. Your instructor will give you a set of "chromosomes" to work with. At his or her discretion, the stages of mitosis may be demonstrated in a large “cell” with a combination of the whole classes’ chromosomes.

 

Before you begin, take stock of your chromosomes. How many chromosomes are there?

 

How many homologous pairs of chromosomes?

 

How can you tell which chromosomes are homologous?

 

How can you distinguish the members of the homologous pair from each other?

 

What differences between the chromosomes are represented by the different colors? That is, what is different about the maternal and paternal chromosomes?

 

 

Procedure

 

In the space below, sketch your chromosomes. You shouldn't take the time to draw each pop bead, but do be sure that homologous pairs can be identified in your drawing.

An example is shown below, with three homologous pairs.

 

 

 

 

Interphase

Before cell division begins, each chromosome must replicate. This event happens while the chromosomes are still in their long, stringy, uncoiled state, so when interphase is viewed under the microscope, the chromosomes are not even identifiable.

 

Using more pop beads, make an exact copy of each of your chromosomes. At this point the chromosome is doubled, with an attachment point called the centromere. Each strand of the doubled chromosome is called a chromatid. Because they are identical, chromatids that are attached to each other are called sister chromatids. Sketch your chromosomes below.

 

 

How many chromosomes are there now? (As long as the chromatids are attached, the structure is considered one chromosome.)

 

How many homologous pairs of chromosomes?

 

 

How many (sister) chromatids?

 

 

Besides replication of the chromosomes, what else should happen during interphase?

(Hint: The entire cell is going to divide in half, not just the nucleus.)

 

After interphase, cell division is a matter of sorting out the cell contents into two new cells. How the chromosomes are sorted is of special interest. By taking a "snapshot" of the process at several points, we can understand the mechanism of this division. Traditionally, the "snapshot" phases of mitosis are called prophase, metaphase, anaphase, and telophase, but keep in mind that cell division is a continuous process.

 

Prophase

It is during prophase that the chromosomes coil more tightly and become visible as distinct structures.  Since we already modeled short, thick chromosomes during interphase, there are no changes to our pop bead model. 

 

By the end of prophase, the nuclear envelope disintegrates, enabling the chromosomes to be snared by protein fibers that have grown out of two centrosomes that formed in the cytoplasm.  Together these protein fibers form the spindle, an apparatus that will eventually allow the sister chromatids to separate from each other.

 

 

 

Metaphase

By metaphase the two centrosomes have moved to opposite ends of the cell with their protein fibers attached, and many of these protein fibers are also attached to the centromere region of a chromosome. One sister chromatid of each chromosome is connected to each pole by this spindle apparatus.

 

The distinctive characteristic of metaphase is the alignment of the chromosomes in a plane through the center of the cell. (Only the centromeres are aligned.) Place your chromosome models in this position and sketch metaphase in the circle below.

 

 

 

 

Based on what you know about the outcome of mitosis, can you guess what's going to happen next?

 

 

 

Anaphase

During anaphase the centromeres joining the sister chromatids separate and the chromatids-now called chromosomes-move along the spindle fibers to opposite poles. The number of chromosomes in the cell is briefly double as the cell is prepared for the final phase of division.

 

Place your chromosome models in the anaphase positions and sketch them in the circle below

 

 

Telophase and Cytokinesis

Now that the chromatids have separated, the division of chromosomes is complete. All that remains is to reconstruct the nuclear envelopes and divide the cytoplasm between the two new cells. These processes will be finished by the end of telophase, and the chromosomes will resume their typical long, stringy form.

 

Place your chromosomes in the telophase positions and sketch them below.

 

 

 

As the chromosomes go through telophase, the division of the cytoplasm, called cytokinesis, is also completed.  The daughter cells separate from each other.  Draw the chromosomes in the daughter cells below.  

 

 

 

 

 

How many chromosomes are there in each new cell?

 

 

How many homologous pairs of chromosomes in each new cell?

 

 

What is the overall result of mitosis?

 

After seeing the phases of mitosis, you may be wondering why cell division is such an elaborate process. Once interphase is complete, why not cut to the chase--telophase? Mitosis has to be carefully choreographed so that each daughter cell has the same number and type of chromosomes-that is, so the division of chromosomes is absolutely equal. Did you ever share a bag of M&Ms with a sibling or friend by carefully counting out and dividing one color at a time? The purpose was to be sure that the distribution was exactly equal, and mitosis has the same purpose regarding chromosomes. On the other hand, you were probably willing to divide a Snickers bar down the middle, without bothering to count every last peanut. You made the assumption that both halves were more or less equal. That's how the cytoplasm and included organelles are divided.

Activity B: Viewing Mitosis in Onion Root Tip Cells

 

Plants have regions of cell division where growth occurs. One such region is the root tip, whose growth enables the roots to elongate and reach through the soil. Since these tips are fine, threadlike tissues, it is easy to press them flat on a slide to view them under a microscope. Mitosis is concentrated in one particular region of the root tip, called meristematic tissue (see Figure 9.2). After the new cells are produced, they will enlarge and mature into different cell types in the root. To produce these root tips, leeks, members of the onion family, were placed in water. You will work with them after several days of root growth.  

 

MATERIALS

• Onion (Allium cepa) or leek root tips

• 9:1 mixture of 2.0% aceto-orcein and 1 N HCl,

• watch glasses, alcohol lamps

• microscope slides, cover slips

• paper toweling

• pencils with erasers

• microscopes.

 

Procedure Staining  of Onion Root Tip Squash (1)

1. Go to the growing onions and, using a razor or scalpel, carefully cut off about l cm of a lateral root (from a main root) of the onion.
2. Place this root tip in a watch glass containing a 9:1 mixture of'  2% aceto-orcein and 1 N HCl. Heat the watch glass and contents gently over an

alcohol flame for a few seconds. Carefully    Zone of (HOT!) remove the watch glass from the flame       cell

before the appearance of fumes from the                  division

solution; you will see the solution has evaporated slightly at the periphery.

3. Leave the root tips in this mixture for approximately 15-20 minutes.

  

While you are waiting, do Activity C (below) : Mitotic Sequence in Cell Models

 

4. Transfer a root tip to a microscope slide, remove all but the terminal (end tip) portion (about 1.5 mm), and cover with a fresh drop of 2% aceto-orcein.
5. Cover the root tip with a cover glass, and place paper towels over the cover glass and slide so as to prevent staining of your hands. Now apply uniform pressure on the cover glass, through the toweling, using the eraser end of a pencil. After most of the stain has seeped out onto the toweling, continue squashing without the toweling until the cells are compressed into a monolayer.
6. Examine your preparation under the microscope (low power) to make sure your staining is good and that you have squashed the root tip sufficiently. To be satisfactory, you should observe a monolayer of tissue. If there are still multilayers of cells, squash (gently) again.
7. Observe your slide for mitotic figures. Draw several phases of mitosis that you can clearly see in the space provided.

 

(1) This exercise prepared by Mary Theresa Ortiz, Ph.D., Anthea M. Stavroulakis, Ph.D., Peter Lanzetta, Ph.D., and Peter Pilchman, Ph.D.

Plant Cell Mitosis Drawings

 

Interphase		Prophase
Metaphase		Anaphase
Telophase		Cytokinesis

 

Activity C:  Determining the Proper Seqeunce of Mitosis in Cell Models

 

Remember that dyeing cells kills them. Observing stained cells (with their lovely chromosomes) is a static phenomenon, that is, nothing is moving! Imagine yourself as Walther Flemming in the 1870's being the first person ever to see a microscopic field of stained, growing cells from animal tissue. What would be the correct sequence in which to place the cells to correctly establish the steps in cell division?

 

The demonstration table has models of plant and animal cells in the different stages of mitosis. With your working group, line up the models in what you believe to be the correct sequence to demonstrate cell division, or mitosis. Ask you instructor to verify that your sequence is correct.

 

Return to your stained chromosomes.

 

Flemming named the process of cell division "mitosis" from a Greek word for thread because the threadlike chromosomes were so characteristically seen during this process. He also named the "aster". Interestingly, Flemming did not see the genetic significance of this process he had discovered. Although Gregor Mendel had already completed and published his work, Flemming was unaware of it. It would be 20 years later when the Dutch botanist, Hugo De Vries (1884-1935) rediscovered Mendel that Flemming's work would reach its full flowering in that it would provide the physical basis for the movement of the genes that Mendel had worked out through his empirical experiments with garden peas. It is also heartening to note that the German botanist, Karl Correns, and the Austrian botanist, Erich Tschermak both, along with De Vries, independently and all unknown to each other, worked out the basic laws of inheritance in 1900. And each, in researching the scientific literature, came upon the work of Gregor Mendel and were stunned that "their" work had already been done a generation earlier. And all three with the greatest humility acknowledged Mendel as the originator of the ideas, and their work as verifying that original!

 

Continuing historical developments included the theoretical suggestion by the German biologist, August Weismann (1834-1914) that the chromosomes contained the hereditary information. The German botanist, Eduard Strasburger (1844-1912), showed that the sex cells (eggs and sperm) contain 1/2 the number of chromosomes as other body cells, a finding that strongly supported a genetic role for the chromosomes. Strasburger also coined the words "cytoplasm" and "nucleoplasm". And the Belgian cytologist, Edouard van Beneden (1846-1910) showed that the chromosome number was constant in the various body cells and that the chromosome number was characteristic for each species of living organism.

 

If you have time, or at home:

Observe slides or photographs in your textbook of animal cell mitosis (whitefish).  Compare to your observations of the onion (plant) cell mitosis.  Note differences in shape of the cell, the aster, differences in cytokinesis.

 

Observe cell division on the Internet. Observe mitosis in both animal cells and plant cells. Your observations of these "time lapse" motion pictures gives a dynamic quality to mitosis that the 19th century cytologists could only have dreamed about.

 

At the request of your instructor:  

HOMEWORK: to be handed in and graded. Using a hypothetical cell with a diploid number of 4, illustrate mitosis, including all detailed movements of the chromosomes through all the mitotic phases. The drawings can all fit on one side of a page, but they should be large enough to clearly illustrate what is happening to the chromosomes. NEATNESS COUNTS. Completely label each figure of IPMAT. Your instructor will provide clarification to these instructions if you are puzzled in any way.