BSC 1010c

BSC1010L

Lab # 9 Mitosis and Meiosis

OBJECTIVES:

A

fter completing this lab exercise, you should be able to:

1. Describe the activities of chromosomes in the cell cycle including all the stages of mitosis and meiosis.

2. Identify the stages of mitosis in the onion root tip and whitefish blastula cells.

3. Describe the differences in mitosis and cytokinesis in plant and animal cells.

4. Describe the differences between mitosis and meiosis.

5. Prepare and observe giant salivary gland chromosomes in Drosophila.

6. Observe human chromosomes in leukocytes and demonstrate human karyotyping.

7. Explain crossing over and describe how this can bring about different arrangements of ascospores in the fungus Sordaria.

INTRODUCTION:

A

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

There are two types of nuclear division: mitosis and meiosis. Mitosis usually results in the production of two daughter cells, which are genetically identical to each other and to the parent cell. Formation of an adult organism from a fertilized egg, asexual reproduction, regeneration, and maintenance or repair of body parts are all accomplished by mitotic cell division. Meiosis, which occurs during the formation of gametes, reduces the chromosome number in daughter cells to half that of the parent cell. Gametes in animals and spores in plants are both produced by meiotic division. The egg and sperm, though usually unequal in size, donate an equal number of chromosomes to the developing organism: each contributes the haploid number of chromosomes. The resulting zygote therefore contains a diploid number of chromosomes. In humans, for example, the zygote contains the full complement of 46 chromosomes while the egg and sperm contain the haploid number of 23 chromosomes.

In this lab, you and your partners will first explore mitosis in onion root tip and whitefish blastula. Then, you will observe giant chromosomes in the salivary glands of fruit fly larva, and prepare slides of human chromosomes. Finally, your team will study meiosis and crossing over in two strains of the fungus Sordaria.

Exercise 9.1 The Cell Cycle and Mitosis

The complex series of events that encompasses the life span of an actively dividing cell is called the cell cycle. It includes an interphase during which the cell appears to be outwardly quiet and an M phase (for mitosis) during which the cell is actively dividing.

Even though it is a continuous process, the cell cycle can be divided into several stages (see Figure 9.1). After a cell divides, it enters interphase, which consists of three stages: G1, S, and G2. Interphase is frequently referred to as a resting stage, but the cells are actually quite active and are preparing for the next division.

During interphase, DNA, with its chromosomal proteins, exist in a highly uncoiled state. Thus, when cells are stained during interphase, distinct chromosome structures are not visible. Chromosomes appear, instead, as a granular material called chromatin within the nucleus.

Events during G1: (G for Gap). The cell approximately doubles in size and its organelles and enzymes double in number. Centrioles also begin to divide during G1. Cells that normally do not divide remain in G1. Actually some cells like muscle and nerve cells will never divide again and are said to be in G0 while others such as liver cells have the potential to divide again if the liver is damaged and pass out of G1 and into S.

Research indicates that a restriction point, R, exist in G1. Passing beyond this point seems to require a certain concentration of a particular protein (MPF) produced in small quantities. The R point might also be affected by cell density; when enough cells are present in a given area division stops. One of the characteristics of cancer cells is that they seem to disregard the signals that restrict division in normal cells. Thus, discovering the exact mechanism of the R point is a subject of intense interest and research.

Events of S: Before the S phase, each chromosome consists of a double-stranded helix of DNA. During the S phase, the two strands of the DNA helix unwind, separate, and duplicate by the process of replication. By the end of the S phase, each chromosome is composed of two helices of DNA called chromatids, joined at a region of the chromosome called the centromere. Distinct chromosomes are not yet visible during this phase. The DNA molecules are intact, but are largely uncoiled and dispersed as chromatin. Chromosomal proteins, also synthesized during S phase, will eventually associate with DNA to help it coil into tightly packed chromosomes prior to the beginning of mitosis.

Events of G2: During this phase structures directly involved in cell division are synthesized. Spindle fibers begin to assemble. These will become attached to chromosomes and guide their movement during mitosis. In animal cells, a pair of centrioles divides to form two pairs of centrioles. These will also play a role in the movement of chromosomes during the mitotic process. Cells of higher plants have spindle fibers but usually lack centrioles.

A. Mitosis in Plant Cells

The onion root tip is one of the most widely used materials for the study of mitosis because it is readily available, preparation of the dividing cells is easy and the chromosomes are large and few in number. The root tips are regions of active cell division so chances are pretty good that in root tip specimens, you can find every stage of mitosis.

As you examine prepared and student-made slides of Allium and later of whitefish blastulas, refer to the following descriptions and diagrams (Figure 9.2(b), page 9-8) and use your textbook in order to learn more about each stage.

Interphase So named because early scientists thought it was resting phase. In reality, the cell is actively undergoing synthesis of DNA and proteins and preparing for mitosis. Most of the cells you observe will be in this mode.

Prophase It is the longest of the four stages of mitosis. The chromosomes shorten and thicken and become progressively visible. The double stranded chromosomes now turned chromatids are clearly seen. The spindle forms and the centrioles move to opposite poles. By the end of prophase, the nuclear membrane and nucleolus have disappeared.

Metaphase This phase is relatively quick and features the chromatids attaching to the mitotic spindle at the centromeres. By the end of metaphase the chromatids have lined up along the metaphase plate.

Anaphase The most rapid stage of mitosis features the splitting of the centromeres and the pulling of the chromatids by the microtubules to opposite poles of the cell. This stage can be recognized in the onion cell by two groups of V-shaped chromosomes on opposite sides of the cell.

Telophase This stage is nearly the reverse of prophase. The chromosomes at the poles begin to uncoil and resume the chromatin form. The spindle fibers disappear and a nuclear membrane forms around each chromatin mass. Nucleoli also appear in each of the daughter nuclei.

Materials:

Clean microscope slides and cover slips

Onion bulb with roots

Dissecting needle and razor blade

Fixative solution (HC1-95% ETOH)

Preservative solution (HAc-70% ETOH)

Aceto-orcein stain

Prepared slides of Allium (Onion root tip) and whitefish blastulas

Procedure

1.Read pages 9-4 through 9-6 about chromosomes and the discoveries that established the chromosome theory of inheritance.

2.Follow steps 1 through 26 on pages 9-7 and 9-8 in order to make your own onion root tip slides. Go to Step 4 on page 9-11 while the slide is being stained.

(Pages 9-4 to 9-10 are from Prof. Bernard Fritzes original write-up)

LABORATORY INVESTIGATION NINE (D: Cytogenetics, C: Chromosomes, Copyright 1993 by Bernard H. Fritze, INTELPROP)

A. Chromosomes are the Microcassettes of Genetic Instructions Parents Pass to their Progeny in Cells.

Survival of the fittest individuals in each generation in the struggle for life is a necessary but insufficient criterion of success. To survive and then parent the new generation, to be a participant in perpetuating the species, defines fitness and success in the context of evolutionary change.

The most singular property that sets a living being apart from all inanimate objects is the capacity to parent, to create copies of one self. This essential character exists even in the smallest globules capable of maintaining the living state, cells. Though measured in thousandths a dimes thinness (micrometers), cells reproduce by becoming two. Cell reproduction is no blob of bubble gum dividing in half as some imagine.

Reproduction begins within the nucleus, the cells library of building and operation instructions recorded digitally along the double ribbon molecule, deoxyribonucleic acid (DNA). All the recording tape is first duplicated. The tape is then packaged with protein as a cassette, the chromosome, and each kind of organism has a specific and countable number. Human body cells have 46 chromosomes, cats 38, corn 20, fruit flies 8, a horse roundworm 4, etc. This is before they are duplicated.

To survive each newly divided cell must have a complete library of operating instructions, meaning all the chromosomes. With all the chromosomes now in duplicate the next sequence of phases is a very precise separation of the twin copies from one another and their assembly into two identical sets in each forming nucleus. The remainder of the cell, the protoplasm, is then divided approximately in half. This is cytokinesis, the nuclear process being called mitosis.

Obviously, large multi-cellular creatures, such as whales or even worms, cannot multiply by dividing. Here, too, the process of reproduction is essentially cellular with the formation of specialized sex cells (gametes), egg and sperm, in special organs (gonads), ovaries and testes.

In the gonads (sex organs) cells that will produce gametes (sex cells) duplicate their chromosomes. Matching chromosomes named homologues of maternal and paternal origin mysteriously find each other and align lengthwise, an event called synapsis. This event only occurs during this kind of division, meiosis, not during ordinary mitosis. Since each homologue is twinned, a bundle of four chromatids, a tetrad, forms.

Homologous (non-sister) chromatids often cross-splice equivalent segments,so-called crossing over. New gene sequences are formed. Each tetrad of four chromatids is independently maneuvered to the cells equatorial plane, creating a large variety of random assortments in the many cells engaged in the process.

The first segregation parts the twinned homologues from each other and the second separates the identical twin chromatids.

Crossing over re-assorts the genes along the chromosomes length, random alignment at the cells equator creates myriad chromosome assortments and two divisions reduces their number to half that of body cells.

Fertilization of egg by sperm restores the double chromosome number in the first body cell of the new life, the zygote or fertilized egg. But more than, that each new individual has a unique assortment of genetic instructions pooled by the union of sex cells produced by the special nuclear phases of meiosis.

And then the random circumstances of the environment, Nature, select those individuals with the best combination of genetic traits to survive and parent another generation. Generation after generation this repeating process has caused biological change, which is evolution.

B. Discoveries Establishing the Chromosomes Theory of Inheritance.

1665 Hooke observes microscopically the porous construction of various plant tissues and names the tiny boxes cells.

1833 Brown discovers the nucleus as a consistent organelle in a great variety of plant cells.

1839 Schwann writes, The most frequent and important basis for recognizing the existence of a cell (plant or animal) is the presenceof the nucleus.

1839 Von Mohl reports cell division is easily seen in plant root tips.

1854 Newport is the first to prove the sperm cell penetrates the frog egg cell to begin fertilization.

1855 Virchow states that new cells originate only from older cells and in no other way.

1866 Mendels cross breeding experiments with peas establish that factors determining inheritance are paired particles that segregate into egg and sperm and recombine at fertilization. At the time these particles were hypothetical abstractions.

1866 Haekel believes the nucleus controls inheritance.

1873 Schneider in studies of flatworm embryos cells is first to report the appearance of stainable threads (chromosomes) in place of the nucleus and their segregation into each of the twin cells resulting from the division.

1876 Hertwig is first to observe the fusion of the sperm nucleus with the egg nucleus 15 minutes after penetration in sea urchin fertilization and the appearance of carmine stainable filaments (chromosomes) during the following first cell division.

1882 Flemming in studies of living and stained salamander embryo cells details the condensation, duplication count and segregation of chromosomes. He names the process mitosis after the Greek word for strings or threads.

1884 Hertwig, Weismann and others conclude inheritance is passed on by the chromosomes of the nucleus.

1887 Weismann theorizes chromosomes occurring as equal parental sets (male and female) whose number must be halved before the doubling of their count that occurs at fertilization.

1887 Van Beneden establishes the chromosome number in all body cells is the same but varies with each species. He shows the number doubling before egg and sperm cells are formed. Two subsequent cell divisions reduce the number to half.

1888 Boveri in studies of an intestinal roundworm with only 4 chromosomes observes the number reduced to 2 in the formation of egg and sperm (meiosis), paired again at fertilization, duplicated and segregated at the first division (mitosis) of the embryo.

1895 Wilson theorizes that stainable chromatin in the nucleus is identical to nuclein, a compound first isolated by Meischer in 1869, and that inheritance might be caused by a specific chemical.

1902 Sutton correlates in grasshopper sperm formation the matching of maternal and paternal chromosomes and their random segregation with Mendels rediscovered paper on the duplicate nature and segregation of the hypothetical elements that control heredity.

1909 Janssens studying the synapsis (pairing) of homologous (matched) chromosomes during meiosis suggests they might break and rejoin, a process called crossing over.

1910 Morgan locates in fruit flies the gene for white eye color on the X chromosomes, a sex chromosome because it determines gender.

1913 Sturtevant maps five gene loci (locations) on the X chromosome of fruit flies based on the frequency of crossing over, fewer if the genes are close together, more if far apart.

1931 McClintock and Creighton prove experimentally crossing over occurs in corn among the 4 chromatids of chromosome 9.

1931 Stern using abnormal X chromosomes of fruit flies establishes microscopically the reality of crossing over.

C. Mitosis and Cytokinesis are Observed in Onion Root Tip Cells Using Squash Technique.

1.Use cleanser at the sink to clean 2 microscope slides. Both will be processed together.

2.Lift the onion bulblet and foam float from the water in the plastic glass. With your fingers pluck an entire root from its base. Keep track of the tapered tip from the torn base end.

3.Place a white root on each of the 2 cleaned slides over the black surface of your desk. Do not allow the root cells to dry out during the rest of this procedure.

4.With a sharp blade cut off 2 mm only of the tip no more. Be sure youve not cut the wrong end. Discard the rest of the root, (2 mm is the size of a capital letter in this text).

5.With the tip of a dissecting needle move the root tip to the far right of the slide. Cover it with several drops of fixative. This instantly kills the cells. They are stopped in the middle of what they are doing, such as dividing.

6.Keep the root tip covered with the fixative for 5 full minutes as it becomes whiter and more opaque. The fixative contains concentrated acid. Keep it all on the slide.

7.At the end of five minutes move the root tip with the dissecting needle to the far left of the slide.

8.Cover it with preservative for 5 minutes.

9.At the end of five minutes use the needle to move the root tip to the center of the slide.

10.Absorb with a piece of paper towel both the fixative and preservative from the ends of the slide, keeping them off your skin.

11.Cover the root tip with the red stain aceto-orcein for the next 25 - 30 minutes, replacing stain as necessary from drying.

12.While the root tips are staining use the time to study the same material (onion) prepared by different technique and using multiple stains rather than one.

13.Obtain from the supply a prepared slide.

14.Hold it over a white background and note the tapered dark streaks, usually 3, under the cover-slip. Each streak is a very thin lengthwise slice of an onion root tapering to the tip.

15.Place the slide on the stage of the microscope and with the scanning objective (lowest power) observe each of the three root tip preparations. Pick the best of the three to study at high powers.

16.Study the tapered tip at scanning power and move up to 100X. Most of the nuclei look like grainy red dots. Find those few that appear to be stringy or show red threads against the green-stained background.

17.Center the cell in the field and swing to high power (40X objective), locking the objective in alignment and increasing the light.

18.Since this root tip has been so thinly sliced, parts of the chromosomes may be missing, making some of the cells difficult to place into the correct stage of division.

19.As many as 95 percent of the cells are in interphase (red dots) and not in the process of mitosis.

20.Cells whose nuclei appear as loose balls of red yarn are in prophase, lasting up to 70 percent of mitosis.

21.Metaphase (chromosomes on the equator), anaphase (two chromosome clumps being pulled apart), and telophase (two stringy red nuclei with a partition wall forming between them) are the rarest but most distinctive phases of mitosis. Here the stringy chromosome threads are most easily distinguished from the grainy blobs of the much more common interphase nuclei.

22.After thirty minutes place each of the two slides on a paper towel. Gently lower a new, unscratched coverslip on to the root tip in the stain. The pressure of the coverslip should squash the root tip into a single layer of cells that appears as a purple-red smudge over the white towel.

23.Fold one end of the paper towel over your slide and apply pressure with your thumb (squash technique) directly over the coverslip. This spreads the root tip cells and absorbs excess stain squeezed from under the coverslip. Removing excess stain makes the stained nuclei and chromosomes clearer.

24.Tapping firmly on the coverslip with a pencil eraser over the squashed root tip should further spread the cells into a single layer. An ideal slide will show no air spaces formed from this treatment.

25.Use the 10X objective to scan the field for those few cells among the many whose nuclei have become strings (chromosomes). Dont use high power to scan the field. Use it only for a closer look at promising prospects. Avoid fields of cells layered upon one another.

26.It is not uncommon to find all the phases of mitosis in a single field even at the highest magnification.

D. The Sequence of 8 Spores in a Sordaria Ascus Reveals Either Segregation or Crossing Over.

(Note: This section is for Exercise 9.4. It is described here to maintain the integrity of Prof. Fritzes original write up.)

1.Observe the Petri plate labeled Sordaria. Keep the lid on. Note the inoculation site of the two parent strains: + is the black spore parent (normal or wild-type) and O is the tan spore parent (mutant).

2.The fungal filaments (hyphae) have grown rapidly over the fresh agar surface, radiating in all directions from the point of inoculation.

3.Within a couple of days or so the hyphae of both fungus strains meet and fertilization occurs as the nuclei fuse.

4.In that zone, roughly the midlines of the plate, tiny pear-shaped reproductive bodies called perithecia form at the agar surface. Their color changes from amber to black as they mature in 5 to 7 days.

5.Within each perithecium are cluster of test tub-like asci, each containing 8 ascospores, the products of a meiosis (plus a mitosis) sequenced in a column in the transparent tube. The row of spores in an ascus can be observed by squashing an ascus cluster (like fingers or bananas) out of a perithecium by applying pressure.

6.Remove the cover of the Sordaria Petri plate and place it on the stereo microscope stage, choosing the best lighting.

7.At the lowest power focus your attention on the zone where the black spore parent and the tan spore parent fungal filaments have grown together, crossed (fused nuclei, fertilized) and have produced the dark pear-shaped perithecia.

8.In the center of a clean slide place a large drop of methyl cellulose.

9.Moving to higher power if needed, use a sharp, angled dissecting needle to select 10 to 15 dark, mature perithecia from a midline of the cross and place them in the methyl cellulose, well separated.

10.Drop a new, unscratched coverslip on the preparation and place the slide on the stereo scope at low power.

11.As you watch, with the tip of the dissecting needle apply pressure on the coverslip to each perithecium, rupturing it and squeezing out the cluster of asci.

12.Over-ripe asci will scatter their spores, but favorable asci will maintain the 8 spores aligned within the tube. Concentrate on those.

13.Scan the slide you have prepared using the 10X objective with the microscope. Ignore asci that are all black or all tan. These are not a cross between wild-type (black) and mutant (tan).

14.An ascus with 4 black spores in one half the tube and 4 tan spores in the other half visualizes the fact of gene segregation (separation of alleles) when sex cells form.

15.Two black, two tan, two black, two tan results from a crossing over between chromatid 2 (paternal homologue) and 3 (maternal homologue).

16.Two tan, two black, two tan, two black is a crossing over of chromatids 1 (paternal) and 4 (maternal).

17.A two tan, 4 black, two tan spore sequence means a crossing over between chromatids 1 (paternal) and 3 (maternal).

18.A two black, 4 tan, two black pattern shows crossing over has occurred between chromatids 2 (paternal) and 4 (maternal).

19.Chromosomes occur as matched pairs (homologues) that may first exchange equivalent segments (crossing over) and then segregate separately into the sex cells, thus reducing their number to half. The half sets are put together and fertilization in an entirely unique assortment in each zygote (first body cell). Such shuffling of the genetic material, genes and chromosomes, generates the variety of life forms from which selection by nature creates the process of evolution, a progression of origination and extinction that still continues even now.

3.After observing the slides you made, make drawings of each stage. Remember to draw only one large cell in each circle.

In the circles below, draw onion cells you observed from your squashed root tips that illustrate the important stages of the cell cycle. Name each stage. Draw one cell in each circle.

KARYOTYPE

A

B

D

E

F

G

SEX

C

KARYOTYPE

AA

BB

DD

EE

FF

GG

SEXSEX

C

1

2

6

54

3

7

8910

11

12

13

1415

19

181716

20

2122

4.Using the high power objective, observe a prepared slide of the onion root tip. Examine a single field in the actively growing region, count and record the phases of the first 50 cells you observe. Record your results in Table 9.1 below. Repeat this count of the two other remaining root tips on your slide. Use Table 9.1 to collect and calculate your results.

Example of Table 9.1 Percent of Cells in Each Phase of the Cell Cycle

Phase

Field 1

Field 2

Field 3

Totals

Percent of Grand Total

Interphase

30

33

27

90

90/120 = 75%

Prophase

10

8

12

30

30/120 = 25%

Table 9.1: Percent of Cells in Each Phase of the Cell Cycle

Cell Stage

Field 1

Field 2

Field 3

Total

% Grand Total (total/grand x 100)

Interphase

Prophase

Metaphase

Anaphase

Telophase

Grand Total

5.The duration of mitosis varies for different tissues in the onion. However, prophase is always the longest phase (1-2 hours) and anaphase is always the shortest (2-10 minutes). Metaphase (5-15 minutes) and telophase (10-30 minutes) are also relatively short in duration. Interphase may range from 12-30 hours. Consider that it takes, on average, 16 hours for the onion root tip cells to complete the cell cycle. You can calculate the amount of time spent in each phase of the cell cycle from the percent of cells in that stage (Note: enter results and calculations in the space below).

Percent of cells in stage x 960 min. = ______ min. of cell cycle spent in stage

(16 hr.) (convert to hours and minutes)

Using the information presented above, calculate the following:

(a) Time spent in Prophase_____hr ___min

Time spent in Metaphase_____hr ___min

Time spent in Anaphase_____hr ___min

Time spent in Telophase_____hr ___min

(b) What was the total time spent in mitosis? ____________

What was the total time spent in interphase? __________

(c) How do your results compare with what is known about the Allium cepa cell cycle?

6.Mitotic Index. In order to get some idea of how many cells are undergoing mitosis at any one time, select at random, the actively dividing zone of two different root tips. In each area, count 100 cells at random and record how many of these cells are undergoing mitosis. Record your data in Table 9.2 and then calculate the percentage of dividing cells and average your results.

Table 9.2: Mitotic Index for Onion Cells

Area 1

Area 2

Average

Total # of Cells

# Cells in Mitosis

Percentage of Cells in Mitosis

B. Mitosis in Animal Cells

Mitosis is easily observed using a prepared slide of whitefish blastula (an early stage of development formed by successive mitotic divisions after the egg has been fertilized by the sperm). Obtain a prepared slide of the whitefish blastula and study it under high power. Identify all stages of mitosis. You may have to examine more than one blastula in order to observe all stages. Draw these stages. Do you notice any differences between mitosis in whitefish blastulas as compared to mitosis in onion root tips? Make a list of these differences.

Exercise 9.2 Giant Chromosomes of Drosophila

What happens if DNA duplicates in preparation for mitosis but cytokinesis does not occur? This actually occurs in some tissues of the fruit fly, Drosophila, during the early stages of larval development. The cells of the salivary glands increase in size but do not divide and as a result, the number of strands making up the chromosome continues to increase. The chromosomes become multi-stranded and are called polytene chromosomes. The DNA content of a polytene chromosome is approximately 1000X greater than the normal DNA content of a regular chromosome.

The many DNA strands of a polytene chromosome condense and fold in the same manner as a single strand of DNA found in chromosomes of other organisms. Highly condensed or folded areas stain darkly and give chromosomes a banded appearance. Human chromosomes also have a banded appearance. The patterns of bands as you will see in Exercise 9.3 can be used to identify genetic abnormalities including chromosomal or gene deletions or rearrangements.

The banded chromosomes are easily seen under low power. This effect is further magnified because homologous chromosomes also pair up. Thus the 8 chromosomes in a fruit fly cell are seen as 4 thick polytene chromosomes.

Materials Needed:

Prepared slide of Drosophila polytene chromosome

Drosophila larva (live)

Clean glass slide and cover slip

Aceto-orcein stain

Dissecting needles and probe

Bottle of 0.7% saline solution

Procedure: (Do only steps one and two. Steps four onwards should be done at the end of the lab if time permits, or if larvae are available.)

1.Obtain a slide of Drosophila polytene chromosomes and observe under low power.

2.Locate a giant polytene chromosome and show it to your partner and the instructor. Draw one chromosome.

a. Do you see any bulges along the length of the chromosome? __________

What do you think they represent? _______________________

b. How many chromosomes does each actually represent? __________________________

Explain your answer:

**************************

3.Obtain a clean slide and place a drop of 0.7% saline toward one end.

4.Use the dissecting microscope and place a Drosophila larva in the saline. Place one needle at the middle of the larva and the other just behind the head (see Figure 9.5) Use a blunt probe to hold the larva at the posterior end and a dissecting needle to remove the head at the first segment behind the mouth.

5.Use a blunt probe to gently press on the anterior part of the larva just behind the third and fourth segments. Roll out and expose the salivary glands. The glands are elongated and semi-transparent. Remove the fat bodies and digestive tube adhering to the pear-shaped glands.

6.Place a drop of aceto-orcein stain next to the drop of saline and with a dissecting needle transfer the glands from the saline to the stain.

7.Stain for 10 minutes. Make sure the drop of stain does not dry up.

8.Place a cover slip on the preparation.

9.Place the slide between the folds of a paper towel and press down on the cover slip firmly. Examine the slide under low power (100X) to locate the chromosomes. Examine the chromosomes to observe the banded pattern. Make a sketch of a banded chromosome in the previous page.

Figure 9.5: Procedure for removing salivary chromosomes from the larva of the fruit fly, Drosophila. The structure of a typical polytene chromosome is also shown.

Exercise 9.3 Human Chromosomes Analysis

INTRODUCTION

The genetic information for all organisms is encoded in the form of one or more DNA molecules. DNA is associated with proteins that function to protect the DNA, facilitate its replication prior to cell division and mediate the expression of the information carried in the DNA nucleotide code; each DNA-protein complex is termed a chromosome. Human chromosomes, like most chromosomes of other eukaryotes (animals, plants, fungi and protists), each contain a single, long, linear molecule of DNA. The chromosomes of these types of organisms are contained in cellular organelles called nuclei. In this lab you will prepare and analyze chromosomes from human tumor cells that have been grown in culture and we will compare them to the standard normal set of human chromosomes with respect to their number, size, proportions and banding patterns.

(The procedure for this section starts on page 24)

CHROMOSOME NUMBER AND HOMOLOG PAIRING

The nuclei of most human cells contain 46 chromosomes: 2 chromosomes are involved in sex determination and are termed sex chromosomes; the other 44 chromosomes are termed autosomes. The autosomes occur in pairs (1-22). Both copies of each chromosome carry the same genes although often different alleles (forms) of those genes; hence, members of a pair are termed homologs. One member of each pair was originally inherited from the individuals mother and the other from the individuals father; cells that contain two sets of chromosomes are referred to as diploid. The gross morphology of the chromosomes of a given pair is generally identical although they differ at the level of nucleotide sequence. (See Figure 9.6 for the standard human chromosome map or karyotype) The two types of sex chromosomes also pair but, in contrast to the autosomal pairs, sex chromosomes are quite different from one another in morphology and carry different genes. The two types of human sex chromosomes are designated X and Y; the X and Y chromosomes have regions of similar nucleotide sequence (termed pseudo-autosomal regions) as well as their distinctive and different genes. The X chromosome carries a large number of genes that are critical to the normal development and function of both females and males. The Y chromosome carries many fewer genes. Among them is the gene that codes for male sex determination. An individual with two X chromosomes is a female, and an individual with an X and a Y is a male because of the critical nature of the genes carried on the X chromosome. All humans must have at least one X chromosome in their karyotype (there are no viable YY individuals).

CHROMOSOME STRUCTURE

CHROMATIN: Eukaryotic chromosomes are made up of a complex of DNA and protein that is termed chromatin. Each human diploid nucleus contains a total of two meters of DNA in the form of 46 individual molecules that are 2 nm in diameter. The DNA is protected by binding with proteins as shown in Figure 9.7. The diagrams in Figure 9.7 are based on images seen with the electron microscope and on the results of biochemical experiments in which chromatin, extracted from cells was analyzed. During interphase, the period between cell divisions, the DNA-protein complex is relatively extended. The long fragile fibers of DNA are protected by complexing with the histone proteins to form nucleosomes. The beads-on-string structure of the nucleosomes is condensed to form a solenoid (a thicker fiber) that, in turn is locally condensed in the form of loops. Experimental evidence indicates that during interphase these loops are probably stabilized by binding to the matrix proteins present in the nuclei. Interphase chromosomes are organized as shown in the first four levels of Figure 9.7. In living cells, chromosomes at this stage are relatively diffuse, thin (300 nm), tangled-appearing threads that fill the nuclei of the cells.

Figure 9.6: Human chromosome map with G-bands.

From Barch (Ed.). The ACT Cytogenetics Laboratory Manual.

When cells prepare to divide, their chromosomes are duplicated and later a copy of each chromosome is inherited by each of the daughter cells that result from the division. Somatic (body) cells, such as the cell line we will use in lab, divide by a process termed mitosis (see Figure 9.8). During mitosis, the duplicated chromosomes progressively condense to become relatively compact X-shaped structures. Each has characteristic proportions and banding patterns on its arms if appropriately stained. In Figure 9.8 note particularly the late prophase and metaphase stage chromosomes. In the lab you will see that these chromosomes are each made up of two sister chromatids, which are held together at a constricted region, the centromere. The ends of the chromosome arms are termed telomeres. There is experimental evidence that indicates that the coiled looped structure of a chromosome in a dividing cell (see Figure 9.8) is stabilized by binding of its chromatin fiber to an internal, X-shaped scaffold of protein.

Figure 9.8: Diagram of a cell in interphase and mitosis. Note especially the changes in cell structure during late prophase. (From Avers, Cell Biology.)

CENTROMERES: Human chromosomes, like those of other eukaryotes, differ in total length and centromere position (see Figure 9.9). In the human karyotype, centromeres of several pairs of chromosomes are positioned so that the two arms of the chromosomes are approximately equal in length (chromosomes 1-3, 19 and 20); these chromosomes are designated as metacentric. Others (4-12, 16-18, and X) have a short arm and long arm, these are submetacentric. In the third type of chromosome (13-15, 21, 22 and Y), the centromere is positioned very close to one end so that one arm is very short relative to the other, these are acrocentric. For all types of chromosomes the shorter arms are termed the p (petite) arms and the longer are the q arms. In Figure 9.10, which are the p arms and which are the q arms? How would you classify this chromosome in terms of centromere position?

TELOMERES: Telomeres cap the ends of the chromosome arms. The DNA in these regions consists of a short (six nucleotides) repeated sequence that is added to each end of the chromosomal DNA molecule by an RNA-protein complex termed a telomerase. Because telomerase activity has been found to be present in germ line cells (cells that produce eggs or sperm) but has not been detected in normal somatic cells, it has been suggested that telomeric sequences are normally added to the ends of chromosomal DNA only in germ line cells. Telomeric sequences are thought to function in protecting the informational content of the chromosomes and to be in the same way involved in the ability of the chromosomes to be replicated. During the successive cell divisions that are required to produce an individual organism from a fertilized egg, the telomeric sequences become shorter with each cell division because of the nature of DNA polymerase, the enzyme that mediates DNA replication prior to cell division and because of the lack of telomerase activity in these later stage cells. Thus telomeres may act as replication clocks that are set at fertilization. As the telomeres shorten, the chromosomal DNA may become increasingly vulnerable to damage and in some way that is still unclear the chromosomes may lose their ability to be replicated. When chromosome replication does not take place, cell division is blocked and the cells become senescent. Aging on the level of entire organisms may be the result in part of this cellular senescence. Recent research has found that cells taken directly from malignant tumors and from permanent (transformed) cell lines frequently contain telomerase activity, these findings lend support to the hypothesis that telomere length is related to cell division capability as both of these latter types of cells are able to divide an apparently unlimited number of times (Kim, et al. 1994, Science. 266:2011-2015).

BANDING: Chromosome pairs can be individually identified by using specific staining treatments. Several of the treatments produce characteristic bands on the arms or at the centromeric constrictions. Some recently developed techniques result in painting entire chromosomes of a pair by using fluorescent dyes bonded to DNA sequences that localize to those chromosomes in a process called DNA hybridization. In lab we will analyze G-banded chromosomes from normal human cells as well as preparing our own chromosome spreads from cultured cells. G-banding involves treating the chromosomes lightly with pancreatic protein-digesting enzymes (primarily trypsin and chymotrypsin) and then staining them with Giemsa, a mixture of dyes that stain nucleic acids and proteins. The results resemble the banding patterns in Figure 9.6. Some regions will stain intensely (G-bands or G-dark bands) and other regions will stain much less intensely (G-light bands). Interestingly, the G-dark bands, which are A - T rich, tend to contain the genes that code for cell type specific proteins (such as hemoglobin), while the G-light bands, which are G - C rich, contain the housekeeping genes which are expressed in all cell types.

The localized affinity of the stain for the chromatin is presumably the result of differential distribution of proteins that are associated with the DNA. Partial digestion with pancreatic proteases selectively alters the structure of some chromosomal proteins more than others so that either the DNA in different regions of the chromosomes is exposed by the enzyme treatment and binds the Giemsa stain more or less efficiently, or the proteins themselves are staining differentially. The method is reproducible, indicating that as the chromatin fiber of each chromatid loops and condenses to form a sister chromatid, it does so in a regular orderly fashion. The fact that the banding patterns are consistent seems quite remarkable given the structure of metaphase chromosomes (review the condensed, looped structure of the human metaphase chromosome in Figure 9.7).

CELLS USED FOR CHROMOSOMAL ANALYSIS

Chromosomal analysis requires a population of cells in which a reasonably large proportion is dividing. A variety of different cell types can be obtained from individuals grown in culture (as described below) and used for such an analysis. For fetal diagnosis, cells are obtained by withdrawing a sample of amniotic fluid that contains cells derived from the fetus or by sampling chorionic villi (small finger-like processes on the placenta which are fetal in origin). Chromosomal analysis that is done for children and adults generally involves culturing lymphocytes that are isolated from a blood sample. Cells from tumors and cultured cell lines are also analyzed for chromosomal abnormalities. The HeLa cells we will analyze are human cervical carcinoma cells that have been grown in culture since 1951. These seemingly immortal cells are used in research world-wide and much of what we know about the molecular biology/biochemistry of human cells has been derived from HeLa cell studies. Interestingly, the HeLa cell line was found to be one of the cell lines that have telomerase activity in the research described above. Over the years the HeLa chromosomes, like those of other permanent cell lines, have undergone duplication, deletions and rearrangements and are now quite different from the normal human karyotype. In the lab we will analyze the differences. Why do you think a cell line could survive with grossly abnormal chromosomes whereas an organism can not?

APPLICATIONS FOR KARYOTYPING

The ability to identify individual chromosomes in the karyotype of organisms has been very useful as a medical diagnostic procedure and in basic research in genetics and evolution. One can detect variations from the normal karyotype with respect to chromosome number (a condition referred to as aneuploidy) or with respect to rearrangement of chromosomal regions (deletion, duplication, translocation from one chromosome to another non-homologous chromosome, or inversion within a chromosome).

For most animal species, increasing or decreasing the number of chromosomes or number of copies of genes (as in duplicating or deleting regions of chromosomes) almost always causes lethality early in embryonic development. Exceptions to this generalization for the human species are trisomies (having three copies of a given chromosome) for chromosome 8, 13, 18, or 21. Individuals carrying these trisomies are initially viable but generally are mentally retarded, physically abnormal, and have a shorter life expectancy. Aneuploidy of sex chromosomes in many cases results in viability. Although people with such karyotypes are frequently mentally retarded, physically abnormal and/or infertile, XYY individuals are an exception in that they are generally normal males.

Fetal diagnosis is an important application of karyotyping. It is estimated that more than 50% of human pregnancies end in spontaneous abortion; very frequently before the woman is even aware she is pregnant. More than half of the spontaneously aborted embryos that have been studied have been found to have chromosomal abnormalities. Karyotype analysis is routinely done in situations in which there is increased risk of a fetus with abnormal chromosomes (e.g. a pregnant woman over 40 years of age).

There is also medical interest in more subtle chromosomal rearrangements such as translocations and inversions. Many different types of leukemia and lymphoma (disease of white blood cells and the organs in which they are formed) and several types of solid tumors have been shown to be correlated with specific translocation, inversions and/or deletions of chromosomal regions in the affected cells. There is considerable on-going research seeking to identify and characterize the genes that are in the regions of such rearrangements (especially at the breakpoints) as they are likely to be involved in transforming the cells to their malignant growth pattern.

The ability to identify specific chromosomes has been useful in studies that localize genes to chromosomes (e.g. somatic cell genetics and in situ hybridization methods beyond the scope of our discussion here). This technology is being used in the current international effort to map the human genome.

PREPARATION METHODS: CELLS

CULTURING AND BLOCKING CELLS: Cells from sources such as those described above are grown in culture under carefully controlled conditions that maximize the rate of cell division. Some cell types (e.g. lymphocytes from blood) require chemical stimulation to divide in culture. At a stage when many cells in the culture are dividing, a chemical agent (e.g. colchicines or vinblastine) is added to block cell division at metaphase. Briefly review mitosis in Figure 9.8. What types of cells would accumulate in the culture when metaphase is blocked? Why not block mitosis in early prophase or after anaphase?

SWELLING THE CELLS: Cultured cells, which have been arrested in metaphase, are swollen by treatment with a hypotonic solution. Water from the solution diffuses into the cells, increasing their volume and making them fragile.

FIXING THE CELLS: The swollen cells are treated with an acetic acid/methanol solution that interacts with the proteins of the cells to stabilize cellular structures. Cells are preserved by the fixative and can be stored in a freezer for months with relatively little degradation. Note also that the acetic acid/methanol treatment inactivates any infectious agents that might be in the cells.

PREPARATION METHODS: CHROMOSOME SPREADS

PREPARING THE SLIDES: Our starting material in the lab will be HeLa cells that have been cultured, blocked in metaphase with colchicines, swollen by treatment with a hypotonic solution, and then fixed. We will spread the chromosomes on slides by dropping the cell suspension from a height of approximately 12 inches above the slide. When the fragile swollen cells hit the slide, those that were arrested in late prophase and metaphase will generally burst, releasing their chromosomes. What cellular structure is lacking in these cells? How does this greatly facilitate making chromosome spreads? To increase the likelihood that the chromosomes spread out and do not overlap, we will heat them gently by exhaling warm air on them immediately after dropping them on the slide.

STAINING AND PHOTOGRAPHING THE SLIDES: We will stain the slides with a very simple, rapid method that will produce a purple, uniform staining on the chromosomes. After staining the slides, let them air dry and then examine them with a microscope. Scan your slides at low power to locate areas with good metaphase spreads (chromosomes close together, but with little or no overlap). After finding an area of metaphase spread, proceed to viewing the spread under oil immersion.

PREPARATION METHODS: KARYOTYPING

You will be provided with a photograph of a G-banded, normal human lymphocyte chromosome spread (see worksheet 3). The chromosomes were prepared by the basic method outline in the lab protocol and the chromosome preparation was photographed with a 35 mm camera. (The photographs were obtained from the Cytogenetics Lab at the Medical Center). Use this photo to construct your normal human karyotype. Begin by covering the back of the print with double-sided tape, then cut out, sort and identify the chromosomes using size, centromere position and banding as criteria and arrange them as a karyotype.

Procedure:

1. Practice dropping 2-3 drops of solution (use water here) from a Pasteur pipette onto a slide from a height of about 12 inches. The slide can be flat on the lab bench or held at an angle of up to 60 degrees. If you are holding the slide on the angle, have the drops land on the slide near the label and then run down the slide.

2. When you feel confident that you can hit the slide with reasonably accuracy, remove a slide from the Coplin jar of ethanol (please cover the jar after removing the slide).

3. Dry the slide thoroughly with a Kimwipe and let the instructor know you are ready for the HeLa cells.

4. Draw up the cell suspension with a dry Pasteur pipette (fill only two-thirds of the narrow part of the pipette).

5. Hold your slide at the angle you prefer and from a height of 12 inches, drop the cell suspension onto the slide.

6. After the fluid runs down the slide and before it dries, hold the slide in front of your mouth and gently exhale warm air onto the surface carrying the cells. Do this 3-4 times. What is the effect of warming the chromosome spreads at this stage? What would be the effect of blowing forcefully?

7. Allow the slide to air-dry. Make sure you have labeled it with your initials.

8. Dip the slide in stain #1 for 1 second only, and then dip it again for 1 second only.

9. Drain off the excess stain by holding the slide vertically and touching the end to paper towels; carefully wipe off the back of the slide (do not contact the surface onto which you dropped the cells).

10. Dip the slide in stain #2 for 1 second only, and then dip it again for 1 second only.

11. Rinse off unbound stain with 5 dips in water. Wipe off the back of the slide with a Kimwipe.

12. Allow the slide to air-dry after the staining, then examine it beginning with the low power objective and find an area with a number of good chromosome spreads. The most useful spreads are those in which the chromosomes are close together but not overlapping.

13. Look for metaphase spread using the oil immersion objective in your microscope.

Answer the following questions:

A.Examine the chromosome preparation you have made under oil objective (100X objective). Is there a difference in overall length of the chromosomes among the spreads? ____________ Why would you expect to be such a difference?

B.What prominent cellular structure breaks down during prophase of mitosis?

How is the breakdown of this structure useful for making the chromosome preparations?

C. (a) Construct a karyotype (page 30) of the chromosomes from the normal human cell (worksheet 3) as directed in the protocol. Attach the chromosomes to the underlying template provided. Label the groups on your karyotype and indicate which are metacentric, submetacentric, and acrocentric. Fill in the normal cell data in the table below.

(b) Count the HeLa cell chromosomes on your print (Use worksheet 2 instead). Determine how many are metacentric, submetacentric, and acrocentric. Enter that data on the table below.

Normal Human Somatic Cell

HeLa Cell Results

# of Chromosomes per cell

# of Metacentric Chromosomes per cell

# of Submetacentric Chromosomes per cell

# of Acrocentric Chromosomes per cell

(c) Briefly, discuss any differences you found between the normal and HeLa cell karyotypes in terms of the criteria in the table.

(d) What other types of differences do you think you would probably detect if you compared G-banded chromosomes of both types of cells?

D.Why do you think a cell line such as HeLa could survive with a grossly abnormal karyotype whereas an embryo can not?

Exercise 9.4 Meiosis in Sordaria: A Study in Crossing Over

Sordaria fimicola is a fungus that spends most of its life as a haploid mycelium. When conditions are favorable, cells of filaments of two different mating types fuse. Ultimately the nuclei fuse and 2N (diploid) zygotes are protected within a structure called the perithecium. Each 2N (diploid) zygote undergoes meiosis, and the resulting cells (ascospores) remain aligned. The position of an ascospore within the ascus depends on the orientation of separating chromosomes on the equatorial plate of meiosis I. After meiosis I, each resulting ascospore divides once by mitosis, resulting in 8 ascospores per ascus. This unique sequence of events means that it is easy to detect the occurrence of crossing over involving chromatids carrying alleles that encode for color of spores and mycelia.

Materials Needed:

Petri plate containing hyphae resulting from a cross between black and tan spores

Slides and cover slips

Dropping bottle of methyl cellulose

Procedure:

1. Follow steps 1-19 on page 9-8 and 9-9. Answer questions and record results.

A)How can you tell where a mating between the wild black strain (+) and the mutant tan strain (-) took place?

B) What is an ascus?

C)What is a perithecium?

D)In the table below, record the number of asci with spores all of one color (indicating that the zygote was formed by fusion of cells of the same strain), black and tan spores with crossover absent, and black and tan spores with crossover present.

Table 9.4: Number of asci in each category

All spores of one color (all tan or all black)

Crossover absent

Crossover present

E)What percentage of asci observed resulted from fusion of cells from different strains?

F)What percentage of those resulting from the fusion of different strains demonstrates crossovers?

References:

Abramoff, P. and Thomson, R. 1991. Laboratory Outlines in Biology V. Freeman.

Helms, D. 1994. Biology in the Laboratory. 2nd Ed., Worth Publishers.

Morgan, J. and Carter, M.E. 1993. Investigating Biology. Benjamin/Cummings.

Acknowledgements:

We wish to thank the late Professor B. Fritze of Broward Community College for allowing us to use his Lab ideas and techniques that have been incorporated into this Lab (pp. 9-6 to 9-9). We would also like to thank Professor Rosemond Potter of the University of Chicago for sharing her ideas and techniques involving Human Chromosome Analysis that appears in this Lab (pp. 9-11 to 9-21).

Review Questions

Mitosis and Meiosis

1.What are the stages of the cell cycle?

2.What is karyokinesis?

3.What is cytokinesis?

4.Which types of cells undergo: a) mitosisb) meiosis?

5.What is the a) haploid number, b) diploid number of an organism?

6.What are the stages of Interphase?

7.What are the stages of mitosis?

8.What events occur at each stage of a) interphase, b) mitosis, c) meiosis?

9.Which process a) reduces the chromosome number; b) keeps it the same? (mitosis or meiosis)

10.Which structure(s) is (are) found in plant cells but not in animal cells (or vice versa) during mitosis, meiosis or cytokinesis?

11.Which organism has a polytene chromosome?

12How many chromosomes are found in a somatic human cell?

13.What is the function of the fixative?

14.What is the function of the preservative?

15.What was used to stain the onion root tip?

16.Be able to identify the stages of mitosis/meiosis.

17.Be able to recognize the results of crossing over in Sordaria.

18.What is a perithecia?

19.What is an ascus?

20.What are autosomes and how many do humans have?

21.How many sex chromosomes are there?

22.What is a karyotype?

23.What are telomeres?

24.Which terms describe the position of the centromere?

25.What are the names given to the long and short arms of the chromosomes?

26.Which stain gives the chromosomes their banding pattern?

27.Which bands are rich in G-C / A-T?

28.What are HeLa cells? How are they different from normal cells?

29.Which chemical blocks the formation of the spindle fibers?

30.Which important structure disappears and allows the formation of a metaphase spread?

Worksheet 1

REPRESENTATIVE FIELD WITH METAPHASE SPREADS

(Enlarged Picture Taken Under 10X Objective)

a. metaphase b. interphase nucleus

Worksheet 2

METAPHASE SPREAD (HeLa Cells)

KARYOTYPE (46 chromosomes, XY)

Worksheet 3.

METAPHASE SPREAD (Normal cell)

Note: Use this worksheet to do the karyotype.

LAB REPORT #9

Date: ___________________

Name(s) :____________________________________________________

MITOSIS AND MEIOSIS

Exercise 9.1 The Cell Cycle and Mitosis

1. In the circles below, draw onion cells you observed from your squashed root tips that illustrate the important stages of the cell cycle. Name each stage. Draw one cell in each circle.

2. Table 9.1: Percent of Cells in Each Phase of the Cell Cycle

Cell Stage

Field 1

Field 2

Field 3

Total

% Grand Total (total/grand x 100)

Interphase

Prophase

Metaphase

Anaphase

Telophase

Grand Total

3. Using the information in page 9-4, calculate the following:

(a) Time spent in Prophase_____hr ______min

Time spent in Metaphase_____hr ______min

Time spent in Anaphase_____hr ______min

Time spent in Telophase_____hr ______min

(b) What was the total time spent in mitosis? ____________

What was the total time spent in interphase? __________

(c) How do your results compare with what is known about the Allium cepa cell cycle?

4. Mitotic Index see page 9-5 to complete the table below:

Table 9.2: Mitotic Index for Onion Cells

Area 1

Area 2

Average

Total # of Cells

# Cells in Mitosis

Percentage of Cells in Mitosis

5. Mitosis in Animal Cells List three (3) differences between mitosis in animal cells and mitosis in plant cells.

(a) ___________________________________________________

(b) ___________________________________________________

(c) ___________________________________________________

Exercise 9.2 Giant Chromosomes of Drosophila

1. Do you see any bulges along the length of the chromosome? __________ What do you think they represent? __________________________

2. How many chromosomes do you see? __________________________

(Note: Homologous chromosomes are paired along their entire length, so what appears as a chromosome is actually two).

Exercise 9.3 Human Chromosome Analysis

1. Examine the chromosome preparation you have made under oil (100X objective). Is there a difference in overall length of the chromosomes among the spreads? ____________ Why would you expect to be such a difference?

2. What prominent cellular structure breaks down during prophase of mitosis?

How is the breakdown of this structure useful for making the chromosome preparations?

3. (a) Construct a karyotype(see back of this page) of the chromosomes from the normal human cell (worksheet 3) as directed in the protocol. Attach the chromosomes to the last page of the Lab Report using the underlying template as a guideline. Label the groups on your karyotype and indicate which are metacentric, submetacentric, and acrocentric. Fill in the normal cell data in the table below.

(b) Count the HeLa cell chromosomes on your print. Determine how many are metacentric, submetacentric, and acrocentric. Enter that data on the table below.

Normal Human Somatic Cell

HeLa Cell Results

# of Chromosomes per cell

# of Metacentric Chromosomes per cell

# of Submetacentric Chromosomes per cell

# of Acrocentric Chromosomes per cell

(c) Briefly, discuss any differences you found between the normal and HeLa cell karyotypes in terms of the criteria in the table.

(d) What other types of differences do you think you would probably detect if you compared G-banded chromosomes of both types of cells?

4. Why do you think a cell line such as HeLa could survive with a grossly abnormal karyotype whereas an embryo can not?

Exercise 9.4 Crossing Over in Sordaria

1. How can you tell where a mating between the wild black strain (+) and the mutant tan strain (-) took place?

2. What is an ascus?

3. What is a perithecium?

4. In the table below, record the number of asci with spores all of one color (indicating that the zygote was formed by fusion of cells of the same strain), black and tan spores with crossover absent, and black and tan spores with crossover present.

Table 9.4: Number of asci in each category

All spores of one color (all tan or all black)

Crossover absent

Crossover present

5. What percentage of asci observed resulted from fusion of cells from different strains?

6. What percentage of those resulting from the fusion of different strains demonstrates crossovers?

Worksheet 3.

METAPHASE SPREAD (Normal cell)

Note: Use this worksheet to do the karyotype.

Figure 9.1: Stages of the Cell Cycle.

G1 and G2 stand for the first and second gaps of interphase. S stands for the synthesis of DNA.

Fig. 9.2(a): Mitosis in Plants

Proceed to step 4 on page 11 viewing the prepared onion root slide.

Fig. 9.2(b): Phases of Mitosis

Fig. 9.3: Meiosis in a Mold

Fig. 9.4: Chromosome continuity

Giant Polytene Chromosome

Figure 9.7: Model for the stages in the condensation of DNA (a) as it complexes with proteins to form chromatin (b-e) and metaphase chromosomes (f). From Barch (ed.), THE ACT Cytogenetics Laboratory Manual.

Figure 9.10: Whole mount of human chromosome 12 photographed with a transmission electron microscope. (From Avers, Cell Biology.)

Figure 9.9: Chromosome Classification.

(a) Metacentric (b) Submetacentric (c) Acrocentric (d) telocentric

Giant Polytene Chromosome

Use Worksheet #3 to do this karyotype.

Rev. SUM109-1