Laboratory. Mitosis, Meiosis and Chromosomes

Transcription

1 Laboratory 8 Mitosis, Meiosis and Chromosomes

2 2 Laboratory 8: Mitosis and Meiosis OBJECTIVES After completing this lab you will be able to: 1. Identify the stages of mitosis and meiosis.. 2. Locate the stages of mitosis in onion root tips. 3. Find and describe the stages of meiosis in lily anthers 4. Describe polytene chromosomes is olated from fruit-fly saliva ry glands LAB PREPARATION Before lab, you should do all of the following: 1.Read Chapter 12 and 13 in Campbell (8 th Ed.) 2.Bring personal protective gear : lab coat, safety goggles, and safety gloves. INTRODUCTION Although some genetic information is carried within organelles such as mitochondria and chloroplasts, it is the nucleus that contains the bulk of the cell s genetic information (DNA). The process of mitosis divides this nuclear genetic materi al equally between daughter cells during cell division. The distribution of identical genetic information is brought about by the longitudinal splitting of the chromosomes. A complex spindle apparatus arranges the separation of one whole set of identical chromosomes to each daughter cell. The normal processes of mitosis and cell division are under tight control within the cell. Only through the gradual emancipation of a cell from this tight control does a cell turn cancerous. In other words, cancer or tumor growth are the res ult of unc ontrolled cell division. Thus, before we can understand how cancer arises, it is necessary to understand the process of mitosis. Meiosis, on the other hand, is nucl ear division that gives rise to gametes in higher plants and an imals. This process provides the opportunity for genetic recombinati on, leading to genetic variation (diversity) from generation to ge neration. This diversity is very important for the survival of th e species. The process of meiosis consists of two nuclear divisi ons, and results in the number of chromosomes in the ga metes being reduced by half. In the first division, the reduction division, homologous chromosomes are separated randomly between two da ughter cells, giving the haploid chromosome number. During the second division the chromosomes are split at the cent romere, as in m itosis, giving rise to four haploid cells.

3 A. Mitosis in the Onion Root-tip The onion root tip is one of the mo st widely used mate rials for the study of the cell cycle be cause it is readily av ailable, preparation of the dividing cells is easy, and the chromosomes are large and few in number, hence, easier to study than the cells of many other organisms. Since root tips are regions of active cell division, chances are good that in a specimen of such tissues one can find every stage of this process. Resist the temptation to think of the various stages of the cell cycle as a series of indi vidual stages in which one stage is discretely followed by another; th e process is more like a movie. However, it is easier to study the process as a seri es of individual stages (like frames of a movie) that show the important characteristics of each stage. The chromosomes of many plants ar e large and eas y to see. While there are many suitable plants for the study of mitosis, the onion has become a standard in teaching labor atories. Root- tips, with the ir many actively dividing cells, ar e the preferred tissue for examination. Preparation is simple and quick. The tissue is fixed and softened by treatment with acid and heat, both of which serve to break down the cell wall and membra nes. A stain is then applied (toluidine blue in this case), which penetrates the damaged cells and stains the chromosomes. The root-tip is finally squashed by hand in order to spread out the cells and the ir chromosomes for easier viewing. B. Meiosis in the Lily Anther While the cells in soma tic tis sues of plan ts and a nimals r outinely undergo mitosis, only the cells in reproductive or gans under go meiosis. Most animal reproducti ve cells contain numerous, small chromosomes and hence meiosis is dif ficult to observe. The immature anthers of pla nts are of ten used to illustrate meiosis because the cells are lar ge, and relatively few chromosomes are present. Because the developing microspores (pollen grains) usually divide synchronously, many cells in the same phase of meiosis are often visible. Unfortunately, the preparation of anthers for examination of meiosis is difficult and time consuming, requiring embedding of the tissue, staining, and the use of a microtome for sectioning. Because there is insufficient time in this laborat ory to go through these procedures, you will examine prepared slides obtained from a biological supply house. C. Giant Chromosomes of Drosophila Without doubt, geneticists have proclaimed the fruit fly Drosophila melanogaster the animal of choice for ge netic stud ies. While the reasons for this choice are numer ous (e.g., they are easily cultured Laboratory 8: Mitosis and Meiosis 3

4 4 Laboratory 8: Mitosis and Meiosis and have a short generation time), perhaps one of the most important reasons is the giant chromosomes that are present in the salivary glands during the fly s larval stag e. These chr omosomes, the result of repeated duplication of the DN A helix, are massive, and clearly show (when properly stained) a banding pattern which geneticists have exploited to explore the genetic basis of morphology, behavior, etc. The c hromosomes a ttain th eir maximum s ize jus t befor e pupation, i.e., late third instar (see Figure 7-1). They then degenerate. The large, multi-stranded, or polyt ene, chromosomes are not limited to the salivary glands but are also present in the gut and Malpighian tubules of larva and the foot pads of adults. However, they reach their largest size in the salivary glands and are most easily examined in this tissue. D. Heredity and Human Disorders Meiosis occurs repeatedly in our human lifetime, as our testes or ovaries produce gametes (sperm or eggs). Almost always, the meiotic spindle distributes chromosomes to daughter cells without error. But occasionally there is an accident, called a nond isjunction, in which the members of a chromosome pair fa il to sepa rate. This results in gametes with an abnormal, aneuploid number of chromosomes. Most embryos resulting from fertilization that involves aneuploid gametes do not proceed very far in develo pment, but are naturally aborted. Some types of aneuploid embryos, however, can survive to birth and beyond. Nondisjunction in meiosis ca n affect either the autosomes (those chromosomes not directly involved in determining sex) or the

5 Laboratory 8: Mitosis and Meiosis 5 sex chromosomes (the pair of chromosomes responsible for determining the gender of an individual). Alterations of chromosome number and structure are associated with a number of serious human disorders, such as Down syndrome (possessing an extra 21st chromo some) and Klinefelter syndrome (a male posses sing an extra X chro mosome). Other human genetic disorders are caused by alteratio ns in a gene or genes that control specific enzymes or metabolic pathways. These genes may be located on either the autosomes or the sex-chromosomes; the latter disorders are said to be sex-li nked and include red-green color blindness and hemophilia. Some genetic disorders are more disabling than others. Genetic a nd biochemical tests on fetal cells and molecules, as well as examin ation of the fetus with ultrasound, can help people make a decision whether to keep or abort the pregnancy.

6 6 Laboratory 8: Mitosis and Meiosis LABORATORY EXPERIMENTS You should work individually for all of today s laboratory exercises. Experiment 1: Mitosis in the Onion Root-tip You must wear personal protective gear (lab coat, gloves, goggles) when working with 1N HCl, as it is a very strong acid. PROCEDURE 1.Remove ONE ENTIRE ROOT from the base of an onion. (DO NOT remove only the bottom port ion of the root because, if you do, the next student will mistakenly remove the top portion that does not contain any mitotic tissue.) 2.Cut the distal 2 mm from the ti p of the root a nd place it on a clean microscope slide. Cover it with 2-3 drops of 1N HCl and gently warm the slide on the sl ide warmer for 2-3 minutes at o C. 3.Remove the HCl from the slide by blotting with a small piece of bibulous paper. Add 2-3 drops of 0.5% aqueous toluidine blue stain and re-heat for 1-2 minutes. 4.Remove the stain by blotting wi th a small piece of bibulous paper, and cover the root-tip with a drop of distilled water. 5.Apply a cover glass, and with a folded Kimwipe around your thumb (to soak up the excess water) squash the root-tip. Apply some force but DO NOT allow the cover glass to slide. 6.Examine the squashed root tip with your compound microscope and look for the various stages of mitosis. The chromosome number of the common onion is 2N = To get some idea of the length of time that a dividing cell spends in each of the mitotic phases, count the number of cells you find in prophase, metaphase, anaphase, and telophase. Do this for about 30 (or more) divi ding cells. Keep in mind that mitosis is a continuous process, not one in which a cell jumps from one phase to the next. As a result, you will probably find

7 some cells that don t fit nicely into any of the four mitotic phases; classify these as best you can. If mitosis in the onion lasts an average of 30 minutes, what is the approximate length of each of its phases? 8.When you have finished examini ng your root tips, discard the cover glass in the glass only box. Rinse & dry the slide, and return it to the slide box for re-use. Laboratory 8: Mitosis and Meiosis 7 MEIOSIS In meiosis, immature or primor dial germ cells under go a reduction from the diploid number to the haploid number of chromosomes and become mature gametes. As a result, meiosis maintains the chromosome.number constant a nd provides genetic variability because of crossing over and the subsequent exchange of genes between chromosomes. Experiment 2: Meiosis in the Lily Anther PROCEDURE In the back of the lab there are eight empty microscopes and a set of prepared slides showing meiosis in the lily anther. Your TA will ask you to find an example of one of the eight phases of meiosis: prophase I, metaphase I, anaphase I, telophase I, prophase II, metaphase II, anaphase II, and telophase II. When you find the phase, place the slide on the appropr iate empty microscope for your classmates to look at. Each prepared slide normally cont ains several sections through the anther, however, not all the sections (sometimes only one!) contain

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9 the phase of meiosis printed on the slide label. Y ou should, therefore, use your text to confir m your phase identification and not the slide label. When looking fo r your assigned pha se, please take only one slide at a ti me, and return that slide before obtaining another. Meiosis in the Lily anther will be stu died a s it occu rs in the development of mature pollen grains of fl owering plants. These pollen grains give rise to male ga metes, which fuse with an egg to produce a zygote. Laboratory 8: Mitosis and Meiosis 9 Before you leave lab, make certain you can identify EACH stage of meiosis. Experiment 3: Examination of the Giant Chromosomes of Drosophila Larvae Obtain a prepared slide of polytene chromoso mes from Drosophila. Examine th e slide first un der low power and observe how the nucleus of each of the cells is co mposed of a coil of giant, banded chromosomes. Choose a cell in which the coil is well spread out and gradually increase the objective le ns magnification to 100X (oil immersion). Make a sketch of the banded chromosomes from one nucleus in your notebook, showing a short segment in detail. Be sure to completely clean off the oil from the 100X objective with lens tissue when you are finished with your observations!

10 10 Laboratory 8: Mitosis and Meiosis a. Interphase The interphase cell, so named because early biologists thought it was in a resting phase, is actively undergoing respiration and the synthesis of DNA, RNA, and protein in preparation for mitosis. b. Prophase During prophase, the DNA, originally in long, thin strands, becomes condensed as a result of coiling an d supercoiling. The nuclear me mbrane begins to break down, and th e chromosomes are distributed throughout the nucleoplasm. During prophase in the onion root tip, the c hromosomes oft en ap pear as a co iled ma ss. Even at t his ea rly s tage, each chromosome has doubled, though th is is difficult to see on a slide. Under very high magnifications, it is possible to see that each chromosome is composed of two separate strands, the sister chromatids. The two sister chromatids are identical in structure, chemistry, and the genetic information they carry because one was replicated (copied) from the original DNA of the other during the last S phase. The sister chromatid s are joined together at a region of attachment called the centromere. Within this region, each chromatid contains a disc-shaped k inetochore. Microtub ules (called kinetochor e microtubules) insert into th e kinetochores and run from them o utward to the two po les o f the c ell. Other pola r microtubules become organized into the spindle fibers. c. Metaphase During early metaphase, some of the polar microtubules break down and new attachments are made between the kinetochore microtubules and tub ules from the opposite p ole. This results in what appear to be rat her ai mless chromosome mov ements, aptly described as dancing chromosomes. As metaphase progresses, a random breaking and reattachment of kinetochore microtubu les to the polar microt ubules of the same or opposite poles occurs until (again randomly) the ki netochore of on e daughter ch romatid is attached to microtubules from one pole, and the kinetochore of the daug hter chromatid is connected to tubules from the opposite pole. Then the polar microtubules pull in such a way th at th e kinetochores become positioned in a region halfway between the poles. This region, which occupies a plane near the center of the cell (and at right angles to the long axis of the spindle fibers), is c alled the metaphase or equatorial plate. The cell is considered to have reached metaphase when the kinetochores of a ll chromosomes have arrived at this equ atorial plate region. At this time the centromeres d ivide in prep aration for sep aration of th e daughter chromatids during the following stage anaphase. d. Anaphase The sister chromatids that make up each chromosome are separated from each other and are pulled by the microtubules to opposite poles of the cell. As the centromeres are pulled apart, the arms of these (now called) daughter chromosomes are passively dragged along. Thus, anaphase in onion cells can be recognized by the twogroups of V-shaped chromosomes on opposite sides of the cell. The sharp end of th e V is oriented toward the pole of the spindle. Reduce the light by adjusting the diaphragm of the microscope, and try to locate any spindle fibers near the center of the cell. They appe ar as very fine lines between the two groups of chromosomes. Anaphase ends when the newly separated chromosomes arrive at opposite poles of the cells. e. Telophase Karyokinesis is completed durin g telophase, and reor ganization of the contents of the two daughter cells (cytokinesis) b egins. It is often difficult to distinguish late anaphase fro m early telophase in the cells of plan ts. During telophase, however, a cell plate, the first indication that cytokinesis is beginning, starts to form as a fine line across the center of th e cell. When co mplete, the cell plate divides the or iginal cell into two d aughter cells. As telophase progresses, the nuclei begin to reor ganize: the chromosomes uncoil and become longer and thinner, the nuclear membrane reforms, and the nucleoli reappear. Mitosis ends with the assembly of two int erphase nuclei, each with one complete set of single-stranded chromosomes. The daughter cells resulting from mitotic division have the same number and kinds of chromosomes (and therefore the same genetic makeup) as the original cell.

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13 Laboratory 8: Mitosis and Meiosis 13 Fill in this form and submit with your Lab Summary. QUESTIONS TO RESEARCH AND ANSWER 1.a) How many pairs o f chromosomes are contained in each human body cell? b)how many genes do scientists chromosomes? estimate ar e loc ated on those 2.a)What do most genes contain instructions for building? b)what functions do these perform? 3.How many pairs of genes are t color? hought to code for human skin 4.The different forms of genes are called. 5.Alleles are thought to arise by the process called. 6.While some mutations have contributed to healthy human diversity, most mutations are. 7.Two examples of diseases caused by recessive alleles are: 8.Diseases caused by recessive genes are relatively (rare, common, lethal) (circle one) because they are masked by the dominant allele. 9.In contrast to diseases caused by recessive alleles, an example of a disease caused by a dominant allele is.

14 14 Laboratory 8: Mitosis and Meiosis 10.In humans, 22 of the 23 ch. romosome pairs are called 11.In humans, the 23rd pair of chro mosomes is c alled. 12.A normal human female has 2 sex chromosomes, while a normal human male has an and a chromosome. 13.Most genes on the sex chromo somes are located on the chromosome. Very few genes are located on the chromosome. 14.An example of a disease caused by an allele carried on the X- chromosome is: 15.Two examples of polygenic diseases are and 16.One of the most common human chromosome disorders, which involves having an extra 21st chromosome, is called 17.Turners Syndrome in females is a result of. 18.An extra X chromosome in males results in, the symptoms of which are usually treatable through and. 19.Most of us carry lethal recessive genes.

15 Laboratory 8: Mitosis and Meiosis 15 LAB SUMMARY Please include the following in your lab summary. 1. Descriptive title 2. Introduction describing purpose and objectives of this lab activity. Also very briefly describe the general approach taken to achieve these objectives. 3. For the first experiment (Mitosis in the Onion Root Tip) include the following: Draw labeled diagrams that illustrate the stages of mitosis in the onion root tip. Table that lists the number of cells observed in different stages of mitosis. Assuming that the length of time spent in each stage is proportional to the number of cells in that stage calculate the duration of each stage of mitosis (assume mitosis takes 30 minutes). A paragraph describing the trends observed. Also include comment whether or not the results made sense and why. Comment on any sources of error. 4. For the second experiment (Meiosis in the Lilium Anther) draw labeled diagrams that illustrate the stages of meiosis. 5. For the third experiment (Giant Chromosomes of Drosophila Larvae) draw a labeled diagram that illustrates the chromosome. 6. Conclusion that summarizes the experiments and interprets the significance of the results. 7. Attach filled-in (with your answers) pages for QUESTIONS TO RESEARCH AND ANSWER on pages 13 & 14 of your lab description.