LABORATORY EXERCISE # 5

LABORATORY EXERCISE # 5

PERPETUATION OF LIFE

(CELL DIVISION AND GENETICS)

PART I – CELL DIVISION

LABORATORY OBJECTIVES FOR PART I – CELL DIVISION

1. Identify the stages and events of plant and animal mitosis.

2. Contrast plant and animal mitosis.

3. Give the major differences and similarities between the processes and results of mitosis and meiosis.

REFERENCE

Textbook: chapters 7 and 8

Photo Atlas: chapter 2, pgs 17-22

INTRODUCTION

In this laboratory exercise you will become familiar with the events that take place during cell divisions. In multicellular organisms cell division results in a variety of cells having different forms and functions. You will also study the developmental stages of meiosis, a unique type of cell division involved in the formation of gametes (sex cells).

I. Mitosis

For a multicellular organism to grow and survive, the cells of which it is composed must be able to reproduce. Cells reproduce by the process of mitosis. The entire process of mitosis is one of constant, integrated change, with one stage leading into another. The end result of mitosis is two cells (clones) with the same chromosome number as the mother cell. Through mitosis organisms grow and mature and replace dead cells and damaged tissue. All forms of asexual reproduction involve the process of mitosis.

Activity:

A. Mitosis of Plant Cells – Allium Root Tip

You will observe the stages and processes of mitosis in Allium (onion) root-tips. Obtain a prepared slide of the onion root tip. Examine one of the root tips under the 4X power of your microscope. Locate the rounded end where most of the dividing cells will be found. For detailed examination, 40X must be used. NOTE: Since each section is very thin, not all of them will be equally good for studying cell division. Be prepared to examine other sections on the slide or even change slides in order to locate and study the series of events described here. Refer to figure 2.5 in the photo atlas for clarification of the stages and events. Many of the cells in your preparation will be in interphase, once known as "the resting stage" between divisions. Locate these cells on your slide. Look for cells with the nuclear membrane in tact containing nuclei with granular chromatin. During this stage the mother cell is not dividing, but duplication of chromatin material (DNA), synthesis of cell organelles, and growth of the cell occurs. For an illustration of the entire cell cycle, refer to figure 2.3 in the photo atlas.

What are the functions of mitosis?

Where in the human body would you expect to find large numbers of cells dividing by mitosis?

Why is the term "resting stage" a poor term to describe cells in interphase?

1. Prophase

During prophase the chromosomes coil and become distinguishable in the nucleus.
The nuclear membrane then breaks down and the chromosomes become distributed randomly throughout the cytoplasm. Each chromosome has now doubled (replicated) and is now composed of two identical strands that will later be pulled apart and become separate chromosomes. Refer to figures 2.4 and 2.5 in your photo atlas.

Why is it necessary that the chromosomes replicate?

2. Metaphase
During metaphase the duplicated chromosomes become arranged near the center of the cell at a region known as the equatorial plate. This arrangement is in preparation for the separation of duplicate chromatids which occur later. Spindle fibers, forming the spindle, become more apparent. Some of these fibers are attached to the chromosomal centromeres and will pull chromosomes to opposite poles of the cell during anaphase. Refer to figure 2.5 in the atlas.

3. Anaphase
At the beginning of anaphase the two members of the previously doubled chromosomes (chromatids) separate and move toward opposite poles (ends) of the cell. This can be recognized by two groups of roughly V-shaped chromosomes on opposite sides of the cell. Since the onion has sixteen chromosomes, it is seldom possible to see all of them at one time. Reduce the light penetrating through the objective of your microscope and try to find the spindle fibers near the center of the cell. (They are often not visible in a study of this kind.) Refer to figure 2.5 in the atlas.

4. Telophase
During telophase the chromatids arrive at each pole, and a cell plate forms across the center of the plant cell, called cytokinesis. When complete the cell plate will divide the original cell into two daughter cells. As telophase progresses the nuclei begin to reorganize and the chromosomes again become distinct. In late telophase the spindle disappears and the nuclear membrane begins to reappear. Refer to figure 2.5 in the atlas.

The two resulting daughter cells have the same number and kind of chromosomes as the mother cell from which they came. Why?

B. Mitosis of Animal Cells -- Whitefish Blastula

You can readily observe mitosis in animal cells by studying a prepared slide of a whitefish blastula, an early developmental stage formed by successive cell divisions following fertilization of the egg by the sperm. The behavior of the chromosomes in animal cells is essentially the same as that observed in plant cells. In whitefish blastula cells, however, the chromosomes are much smaller and more numerous than in the onion root-tip cells. You will note other more important differences:

1. In animals there is a pair of centrioles located at each pole of the spindle.

2. In addition to the fibers of the spindle there are other fibers called aster rays which radiate away from the centrioles.

3. The mother cell is apparently "pinched" in half at the equator as the two daughter cells are separating during telophase. This forms a cleavage furrow. No cell plate is formed in mitosis of animal cells.

Refer to figure 2.7 in the photo atlas for a comparison of all stages and events.

C. Illustration of Mitosis – Pipe Cleaners

Obtain twelve pipe cleaners from your instructor, four each of three different colors. Two of each color should be plain and two of each color should have black dots.

To illustrate metaphase of mitosis twist the two identical pipe cleaners together at one point. You now have six chromosomes, each made up of two chromatids. The two chromosomes (pipecleaners) of the same color make up a pair. The dotted pipecleaner is the homolog of the plain pipecleaner of the same color. The point of contact represents the centromere. Line the six chromosomes up in a single line as in metaphase. The six chromosomes make up two sets (one maternal and one paternal) or 3 pairs.

To illustrate anaphase of mitosis separate the chromatids and move them apart. You now have two cells with six chromosomes each. Each chromatid is now a chromosome.

II. Meiosis

Meiosis is a unique biological event that functions in production of gametes used in sexual reproduction in animals, and production of spores in plants. It not only maintains the chromosome number constant for a species of plants or animals, but provides for genetic variability through independent assortment of pairs of chromosomes and because of "crossing over", an event which allows the exchange of genetic material. In animal meiosis, immature germ cells undergo a "reduction" from the diploid (2n) number of chromosomes characteristic for the species and become mature haploid (1n) gametes. Mature gametes are sperm and eggs in humans and occur in males in the testes and in females in the ovaries or oviducts. In plants, meiosis results in the formation of spores.

Refer to figure 2.5 for photomicrographs that illustrate and describe the stages of meiosis.

Meiosis involves two cellular divisions instead of one, resulting in the formation of four gametes, each with half the original chromosome number. The two cellular divisions are designated as Meiosis I and Meiosis II. Major events are as follows:

A. Meiosis I

1. Chromosomes appear as double structures consisting of two chromatids.

2. Chromosomes carrying similar (but not identical) traits pair up. This pairing is called synapsis and may result in an exchange of genetic material between chromosomes called crossing over. Thus, tetrads of chromatids are formed.

3. Tetrads gather on the equatorial plane.

4. Whole chromosomes (consisting of two chromatids each) move to the opposite poles. At this point, homologous (like) chromosomes are essentially separated into two different new cells. The chromosome number has been halved.

The pole to which each chromosome of the pairs goes is entirely by chance. What is the significance of this?

B. Meiosis II

1. Chromatids, still joined, move to the equatorial plane.

2. Chromatids separate and move to opposite poles.

3. We now have four (4) cells each with half the normal number of chromosomes.

What does homologous mean? What are homologous chromosomes?

Where in the human body would you expect meiosis to occur?

How many chromosomes are in a human somatic cell? How

many chromosomes are in a sperm or egg?

Are the end results of meiosis always gametes?

Explain.

Activity:

Illustration of Meiosis – Pipe Cleaners

You will now demonstrate what happens during Meiosis I and Meiosis II.

1. Meiosis I

Twist identical pipe cleaners together to represent chromosomes consisting of two chromatids. You now have two sets of chromosomes. Now, also, twist together all four pipe cleaners of the same color. Synapsis of chromosome pairs has occurred. Line the three resulting tetrads up together as occurs in metaphase I of Meiosis I.

To illustrate anaphase I of Meiosis I now separate the solid color pipe cleaners from the dotted ones and move the joined chromatids toward opposite poles. (It makes no difference if some dotted "chromosomes" and some plain ones move toward the same pole.) You have now reduced the chromosome number in the resulting cells by one half. Note that the chromosomes in the new cells do not have both members of a pair.

2. Meiosis II

Continuing from above, line up the pipe cleaners (still joined as chromatids) along their respective equatorial planes. In Meiosis II, chromatids separate and move to opposite poles. To illustrate this, now separate the chromatids and move them to opposite poles. The end result is four groups of three chromosomes. This represents four gametes each containing three chromosomes.

Disregarding crossing over, how many different gametes are possible from these six different chromosomes?

PART II -- GENETICS

LABORATORY OBJECTIVES FOR PART II -- GENETICS

To learn about the basic patterns of inheritance. Upon completion of this laboratory exercise, the student will be able to:

1. Understand basic genetic concepts; including segregation, independent assortment, dominance, and codominance.

2. Understand and solve basic single-trait genetic problems.

3. Solve single-trait genetic problems involving human conditions; including sex determination, sex-linkage, and blood groups.

INTRODUCTION

Living organisms exhibit amazing diversity of appearance and function. Even members of a single species, which share common characteristics, differ considerably in the expression of those characteristics. This variability is not haphazard; rather, individuals tend to express unique combinations of those traits observed in their parents. These traits are determined by units of DNA called genes which are located in linear order on the chromosomes. Genetics is the study of how genes are expressed to produce specific characteristics and how genes are transmitted from one generation to the next.

Genetic information is transferred from parents to offspring by sexual reproduction. This process involves the formation of gametes, the egg and sperm, and their union in fertilization. The cells of the human body which form gametes contain twenty-three pairs of chromosomes. Cells which contain pairs of chromosomes are termed diploid cells. The members of each chromosome pair are similar in appearance. They contain genes for the same traits and are referred to as homologous chromosomes. The genes which occupy identical positions on the homologous pair are called alleles.

During meiosis the homologous pairs separate producing gametes containing twenty-three single or unpaired chromosomes. Since the egg and sperm contain one-half the chromosome number of diploid cell, they are said to be haploid cells. The separation of the members of allelic pairs, which occurs during meiosis, is called segregation. Due to segregation, the egg and sperm contain one allele for each trait.

Fertilization, the union of sperm, re-establishes the diploid condition in a single cell, the zygote. The zygote divides mitotically forming daughter cells which also undergo mitosis. Further division and differentiation of the daughter cells results in the trillions of cells which comprise the adult human.

The diploid cells of the individual contain one pair of alleles for each characteristic. If the alleles for a particular trait are similar in their expression, the individual is said to be homozygous for that trait. If the members of an allelic pair are different in expression, the individual is said to be heterozygous for the trait. The pair of alleles for a specific characteristic is termed the genotype and is represented by letter symbols. The actual appearance of the characteristic in the individual, due to the expression of those alleles, is its phenotype. The phenotype consists of a phrase describing the characteristic such as blue-eyed or brown-haired.

In many allelic pairs, one member masks or hides the expression of its alternative and is termed a dominant allele. The allele which is masked or hidden is called a recessive allele. Capital letters are used as symbols to represent dominant alleles and small letters represent recessive alleles. Individuals who are homozygous (AA) or heterozygous (Aa) for a dominant allele express the dominant phenotype; whereas, only those individuals homozygous (aa) for a recessive allele express the recessive phenotype.

In some pairs of alleles, neither member is dominant, but the heterozygote exhibits a distinct phenotype different from that of either homozygote. If the phenotype of the heterozygote is a combination of both homozygotes, the alleles are said to be codominant. The alleles that produce the A and B blood types are codominant, resulting in the AB blood type in heterozygous individuals.

Activity:

A. Monohybrid Cross Illustration -- Toothpicks

Monohybrid crosses are those in which the parents differ with respect to a single pair of alleles. The Punnett square is commonly used to determine the different genotypes and phenotypes which may result from monohybrid crosses. Consider a man and a woman, both heterozygous (Aa) for a dominant trait. Each can produce two different types of gametes, "A" or "a". When a Punnett square with four divisions is constructed and the possible gametes of the parents are placed at the top and to the left of each division, the proportion of genotypes and phenotypes obtained following fertilization can be determined as follows:

Genotype of parents: (female) Aa X (male) Aa

Meiosis (segregation)

Types of gametes: A or a A or a

Fertilization:

Male Gametes

A a

Female A AA Aa

Gametes

a Aa aa

Genotypic ratio: 1 AA : 2 Aa : 1 aa

Phenotypic ratio: 3 dominant : 1 recessive

You will now attempt to reproduce the above ratios by the following simple activity:

1. Select 12 toothpicks of one color, then select 12 toothpicks of a second color. The toothpicks of one color will represent your possibilities for producing gametes with the dominant allele (A) for normal skin pigmentation, the toothpicks of the second color will represent your possibilities for producing gametes with the recessive allele (a) for albino, or lack of pigmentation.

2. Place the 24 toothpicks into a cup. Without looking, and avoiding all bias in selection, draw one toothpick with your left hand. Record it, by letter, as the allele contained in the sperm. Replace the toothpick into the cup. Again, without looking draw one toothpick with your right hand. Record it, by letter, as the allele contained in the egg. Replace the toothpick.

3. You will record your data on Table 1. Repeat the selection of sperm and eggs until the first and second columns of the table are filled (25 times).

4. Determine the genotypes and phenotypes that will result from the sexual fusion of the two gametes and record in the third and fourth columns of the table.

5. Tabulate the totals and the genotypic and phenotypic ratios. Record your calculations on Table 2. Did your totals and ratios come close what you expected?

Table 1: Monohybrid cross

Trial	Sperm	Egg	Genotype	Phenotype

Ex.	a	A	Aa	normal skin
Ex	A	A	AA	normal skin
Ex	a	a	aa	albino

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

Table 2: Monohybrid Cross

Genotype	Number	Genotypic Ratio	Phenotype	Number	Phenotypic Ratio
AA			Normal pigmentation
Aa			Normal pigmentation
aa			Albino

B. Single-trait Genetics Problems.

1. If one parent is homozygous dominant for a trait, that parent's genotype might be GG. If the other parent is homozygous recessive for the same trait, that parent's genotype would be gg.

a. What are the possible genotypes of their offspring?

b. How many different phenotypes might we possibly see in their offspring?

2. If a male is homozygous dominant for a trait, that parent's genotype might be DD. If the female is heterozygous for the same trait, her genotype would be Dd.

a. How many genetically different sperm can the male produce?

b. Using a Punnett square, determine the expected genotypic ratios of their offspring.

c. What are the expected phenotypic ratios?

3. In garden peas, the gene for red flowers (R) is dominant over the gene for white flowers (r). If pollen (cells containing sperm nuclei) from the anther (male reproductive organ) of a homozygous red-flowered plant is added to the pistil (female reproductive organ containing the egg) of a white-flowered plant:

a. What are the expected phenotypic and genotypic ratios in the first generation of offspring? and ________________________.

b. If two members of the first generation of offspring were crossed, what would be the expected phenotypic and genotypic ratios in the next generation?

and _________________________________.

4. White fruit color in squash is due to a dominant allele. Yellow fruit occurs in plants which are homozygous for the recessive allele. If pollen from the anthers of a heterozygous white-fruited plant is placed on the pistil of the yellow-fruited plant; show, using ratios, the genotypes and phenotypes you would expect the seeds from this cross to produce. and _________________________.

5. In man, the allele for normal color (A) is dominant to the allele for albinism (a). A normal man whose father was albino married a normal woman whose mother was albino.

a. What are the chances that their first child will be albino?

b. What are the chances that their second child will be albino?

6. If an albino woman married a normal man, one of whose parents was albino, what would be the chances of their first child being albino?

Human Genetics

You need only to look around at your family and friends to notice that all humans are similar in many ways. It is also very obvious that there is much variation among us. For example, some people are tall and some are short; some are of dark complexion and some are light; some have big noses and some have small; and, some have type A blood and some have type O. Further, you have no doubt noticed that, in general, family members share more features in common than do individuals who are not related. A biologist would explain that you are more likely to share more of the same alleles with relatives than with members of the public at large. But, gene combinations are continually shuffled as members of different families marry and have children.

Human genetics has a popular appeal to many students who may wonder about differences in hair color, blood types or intelligence. Unfortunately, humans are not suitable subjects for most genetics studies because of their generation times are long, families are small, and test matings are prevented by laws and ethics. However, some rather superficial traits can be noted and this laboratory activity examines some of these.

Activity:

Human Genetics Problems

In this lab exercise, you will determine your phenotype and possible genotype for selected human traits. If the trait results from a dominant gene, you will record two capital letters when you are sure you are homozygous dominant (AA). If the trait results from a dominant gene and you cannot be sure you are homozygous or heterozygous, record a capital letter designating the known allele and a dash for the unknown allele (A-). If your phenotype is dominant and one of your parents is recessive for the trait, then you must be heterozygous (Aa) for the trait. Recessive traits which you possess will be recorded by two lower-case letters because you must be homozygous for the trait to be expressed phenotypically.

Please note: Dominant does not connote "good", nor does recessive connote "bad"

1. Sex is perhaps the easiest trait for which you can determine your phenotype because you are either male or female. In Homo sapiens, sex is determined by a pair of chromosomes (not a pair of alleles). Males have one long chromosome designated "X" and a shorter chromosome designated "Y". Females have two long "X" chromosomes. Record your phenotype and genotype (either XX or XY).

2. Examine your ear lobes in a mirror. An ear lobe is said to be free-hanging if it hangs below the point of attachment to the head. It is described as attached if it does not hang below the point of attachment. The allele for free-hanging ear lobes (L) is dominant to its recessive alternative (l), which produces attached lobes. Record your genotype as LL, Ll, or ll. (Hint: Record Ll only if you have free-hanging ear lobes and one parent has attached lobes.) Record your phenotype.

3. Hair whorl direction is an inherited characteristic. Observe the whorled pattern of your hair at the back of your head. The dominant condition (WW or Ww) is hair at the back of the head whorling in a clockwise direction. The recessive condition (ww) has the hair rotating counter-clockwise. Remember, if you are dominant and both parents are dominant, you must record Ww as your genotype.

4. The ability to taste PTC (phenylthiocarbamide) paper is not possessed by all humans. The taste of PTC paper is extremely bitter to PTC tasters who are of genotype TT or Tt. Non-tasters are homozygous recessive tt. Your instructor will give you a strip of PTC paper. Chew it up; working it toward the back of your tongue. CAUTION: DON'T SWALLOW IT. THE CHEMICAL IS HARMLESS; BUT THE HUMAN DIGESTIVE SYSTEM IS NOT DESIGNED TO DIGEST PAPER. If the paper tastes bitter you are a taster. If it tastes like plain tissue paper, you are a non-taster. Record your results.

5. People with the dominant allele (R) have the ability to roll the tongue into a U-shape when the tongue is extended from the mouth. Those with the recessive genotype are unable to roll their tongue. Are you a tongue-roller or a non-roller? Record both your genotype and your phenotype.

6. Mid-digital hair on the middle joint of one or more fingers is a dominant characteristic (M). People with the recessive genotype (mm) lack hair on the second joint of all fingers. Remember, like always, if your phenotype is dominant, you should try to examine your parents to see if one of them is recessive. Record your genotype and your phenotype.

7. Hitch-hiker's thumb, distal hyperextensibility, is the ability to bend the thumb backward so that the terminal joint forms at least a 45% angle with the joint below it. This trait is due to the recessive allele (h) in the homozygous condition. People with one or more dominant alleles (HH or Hh) will lack this ability. Record your genotype and phenotype.

8. Some traits are termed sex-influenced. Sex-influenced alleles are those whose dominance is affected or altered by the sex of the ind