mitosis, meiosis and chromosomes 171l/lab 08 mitosis... · mitosis, meiosis and chromosomes...

Mitosis,Meiosis and

Chromosomes

Laboratory

8

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 PREPARATIONBefore lab, you should do all of the following:

1.Read Chapter 12 and 13 in Campbell (8th Ed.)2.Bring personal protective gear : lab coat, safety goggles, and

safety gloves.

INTRODUCTION Although some genetic information is carried within organelles suchas mitochondria and chloroplasts, it is the nucleus that contains thebulk of the cell’s genetic information (DNA). The process of mitosisdivides this nuclear genetic materi al equally between daughter cellsduring cell division. The distribution of identical genetic informationis brought about by the longitudinal splitting of the chromosomes. Acomplex spindle apparatus arranges the separation of one whole setof identical chromosomes to each daughter cell.

The normal processes of mitosis and cell division are under tightcontrol within the cell. Only through the gradual emancipation of acell 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 tounderstand the process of mitosis.

Meiosis, on the other hand, is nucl ear division that gives rise togametes in higher plants and an imals. This process provides theopportunity for genetic recombinati on, leading to genetic variation(diversity) from generation to ge neration. This diversity is veryimportant for the survival of th e species. The process of meiosisconsists of two nuclear divisi ons, and results in the number ofchromosomes in the ga metes being reduced by half. In the firstdivision, the reduction division, homologous chromosomes areseparated randomly between two da ughter cells, giving the haploidchromosome number. During the second division the chromosomesare split at the cent romere, as in m itosis, giving rise to four haploidcells.

Laboratory 8: Mitosis and Meiosis 3

A. Mitosis in the Onion Root-tip The onion root tip is one of the mo st widely used mate rials for thestudy of the cell cycle be cause it is readily av ailable, preparation ofthe dividing cells is easy, and the chromosomes are large and few innumber, hence, easier to study than the cells of many otherorganisms. Since root tips are regions of active cell division, chancesare good that in a specimen of such tissues one can find every stageof this process. Resist the temptation to think of the various stages ofthe cell cycle as a series of indi vidual stages in which one stage isdiscretely followed by another; th e process is more like a movie.However, it is easier to study the process as a seri es of individualstages (like frames of a movie) that show the importantcharacteristics of each stage.

The chromosomes of many plants ar e large and eas y to see. Whilethere are many suitable plants for the study of mitosis, the onion hasbecome a standard in teaching labor atories. Root- tips, with the irmany actively dividing cells, ar e the preferred tissue forexamination. Preparation is simple and quick. The tissue is fixedand softened by treatment with acid and heat, both of which serve tobreak down the cell wall and membra nes. A stain is then applied(toluidine blue in this case), wh ich penetrates the damaged cells andstains the chromosomes. The root-tip is finally squashed by hand inorder to spread out the cells and the ir chromosomes for easierviewing.

B. Meiosis in the Lily Anther While the cells in soma tic tis sues of plan ts and a nimals r outinelyundergo mitosis, only the cells in reproductive or gans under gomeiosis. Most animal reproducti ve cells contain numerous, smallchromosomes and hence meiosis is dif ficult to observe. Theimmature anthers of pla nts are of ten used to illustrate meiosisbecause the cells are lar ge, and relatively few chromosomes arepresent. Because the developing microspores (pollen grains) usuallydivide synchronously, many cells in the same phase of meiosis areoften visible.

Unfortunately, the preparation of anthers for examination of meiosisis difficult and time consuming, requiring embedding of the tissue,staining, and the use of a microtome for sectioning. Because there isinsufficient time in this laborat ory to go through these procedures,you will examine prepared slides obtained from a biological supplyhouse.

C. Giant Chromosomes of Drosophila Without doubt, geneticists have proclaimed the fruit fly Drosophilamelanogaster the animal of choice for ge netic stud ies. While thereasons for this choice are numer ous (e.g., they are easily cultured


and have a short generation time), perhaps one of the most importantreasons is the giant chromosomes that are present in the salivaryglands during the fly’ s larval stag e. These chr omosomes, the resultof repeated duplication of the DN A helix, are massive, and clearlyshow (when properly stained) a banding pattern which geneticistshave exploited to explore the genetic basis of morphology, behavior,etc. The c hromosomes a ttain th eir maximum s ize jus t befor epupation, i.e., late third instar (see Figure 7-1). They then degenerate.

The large, multi-stranded, or polyt ene, chromosomes are not limitedto the salivary glands but are also present in the gut and Malpighiantubules of larva and the foot pads of adults. However, they reachtheir largest size in the salivary glands and are most easily examinedin this tissue.

D. Heredity and Human DisordersMeiosis occurs repeatedly in our human lifetime, as our testes orovaries produce gametes (sperm or eggs). Almost always, the meioticspindle distributes chromosomes to daughter cells without error. Butoccasionally there is an accident, called a nond isjunction, in whichthe members of a chromosome pair fa il to sepa rate. This results ingametes with an abnormal, aneuploid number of chromosomes. Mostembryos resulting from fertilization that involves aneuploid gametesdo not proceed very far in develo pment, but are naturally aborted.Some types of aneuploid embryos, however, can survive to birth andbeyond. Nondisjunction in meiosis ca n af fect either the autosomes(those chromosomes not directly involved in determining sex) or the


sex chromosomes (the pair of chromosomes responsible fordetermining the gender of an individual).

Alterations of chromosome number and structure are associated witha number of serious human disorders, such as Down syndrome(possessing an extra 21st chromo some) and Klinefelter syndrome (amale posses sing an extra X chro mosome). Other human geneticdisorders are caused by alteratio ns in a gene or genes that controlspecific enzymes or metabolic pathways. These genes may belocated on either the autosomes or the sex-chromosomes; the latterdisorders are said to be sex-li nked and include red-green colorblindness and hemophilia. Some genetic disorders are moredisabling than others. Genetic a nd biochemical tests on fetal cellsand molecules, as well as examin ation of the fetus with ultrasound,can help people make a decision whether to keep or abort thepregnancy.


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.

PROCEDURE1.Remove ONE ENTIRE ROOT from the base of an onion. (DO

NOT remove only the bottom port ion of the root because, ifyou do, the next student will mistakenly remove the top portionthat does not contain any mitotic tissue.)

2.Cut the distal 2 mm from the ti p of the root a nd place it on aclean microscope slide. Cover it with 2-3 drops of 1N HCl andgently warm the slide on the sl ide warmer for 2-3 minutes at60-70oC.

3.Remove the HCl from the slide by blotting with a small pieceof bibulous paper . Add 2-3 drops of 0.5% aqueous toluidineblue 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. Applysome force but DO NOT allow the cover glass to slide.

6.Examine the squashed root tip with your compound microscopeand look for the various stages of mitosis. The chromosomenumber of the common onion is 2N = 16.

7.To get some idea of the length of time that a dividing cellspends in each of the mitotic phases, count the number of cellsyou find in prophase, metaphase, anaphase, and telophase. Dothis for about 30 (or more) divi ding cells. Keep in mind thatmitosis is a continuous process, not one in which a cell jumpsfrom one phase to the next. As a result, you will probably find


some cells that don’ t fit nicely into any of the four mitoticphases; classify these as best you can. If mitosis in the onionlasts an average of 30 minutes, what is the approximate lengthof each of its phases?

8.When you have finished examini ng your root tips, discard thecover glass in the glass only box. Rinse & dry the slide, andreturn it to the slide box for re-use.

MEIOSIS In meiosis, immature or primor dial germ cells under go a reductionfrom the diploid number to the haploid number of chromosomes andbecome mature gametes. As a result, meiosis maintains thechromosome .number constant a nd provides genetic variabilitybecause of crossing over and the subsequent exchange of genesbetween chromosomes.

Experiment 2: Meiosis in the Lily Anther

PROCEDUREIn the back of the lab there are eight empty microscopes and a set ofprepared slides showing meiosis in the lily anther. Your TA will askyou 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 thephase, place the slide on the appropr iate empty microscope for yourclassmates to look at.

Each prepared slide normally cont ains several sections through theanther, however, not all the sections (sometimes only one!) contain


the phase of meiosis printed on the slide label. Y ou should,therefore, use your text to confir m your phase iden tification and notthe slide label. When looking fo r your assigned pha se, please takeonly one slide at a ti me, and return that slide before obtaininganother.

Meiosis in the Lily anther will be stu died a s it occu rs in thedevelopment of mature pollen grains of fl owering plants. Thesepollen grains give rise to male ga metes, which fuse with an egg toproduce a zygote.

Before you leave lab, make certain you can identify EACH stage ofmeiosis.

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 thenucleus of each of the cells is co mposed of a coil of giant, bandedchromosomes. Choose a cell in which the coil is well spread out andgradually increase the objective le ns magnification to 100X (oilimmersion).

Make a sketch of the banded chromosomes from one nucleus in yournotebook, showing a short segment in detail.

Be sure to completely clean off the oil from the 100X objective withlens tissue when you are finished with your observations!


a. Interphase The interphase cell, so named because early biologists thought it was in a resting phase, isactively undergoing respiration and the synthesis of DNA, RNA, and protein in preparationfor mitosis.

b. Prophase During prophase, the DNA, originally in long, thin strands, becomes condensed as a result ofcoiling an d supercoiling. The nuclear me mbrane begins to break down, and th echromosomes are distributed throughout the nucleoplasm. During prophase in the onion roottip, the c hromosomes oft en ap pear as a co iled ma ss. Even at t his ea rly s tage, eachchromosome has doubled, though th is is difficult to see on a slide. Under very highmagnifications, it is possible to see that each chromosome is composed of two separatestrands, 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 originalDNA of the other during the last S phase. The sister chromatid s are joined together at aregion of attachment called the centromere. Within this region, each chromatid contains adisc-shaped k inetochore. Microtub ules (called kinetochor e microtubules) insert into th ekinetochores and run from them o utward to the two po les o f the c ell. Other pola rmicrotubules become organized into the spindle fibers.

c. Metaphase During early metaphase, some of the polar microtubules break down and new attachmentsare made between the kinetochore microtubules and tub ules from the opposite p ole. Thisresults 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 ofkinetochore microtubu les to the polar microt ubules of the same or opposite poles occursuntil (again randomly) the ki netochore of on e daughter ch romatid is attached tomicrotubules from one pole, and the kinetochore of the daug hter chromatid is connected totubules from the opposite pole. Then the polar microtubules pull in such a way th at th ekinetochores become positioned in a region halfway between the poles. This region, whichoccupies a plane near the center of the cell (and at right angles to the long axis of the spindlefibers), is c alled the metaphase or equatorial plate. The cell is considered to have reachedmetaphase when the kinetochores of a ll chromosomes have arrived at this equ atorial plateregion. At this time the centromeres d ivide in prep aration for sep aration of th e daughterchromatids during the following stage–anaphase.

d. Anaphase The sister chromatids that make up each chromosome are separated from each other and arepulled 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 onopposite 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 spindlefibers near the center of the cell. They appe ar as very fine lines between the two groups ofchromosomes. Anaphase ends when the newly separated chromosomes arrive at oppositepoles of the cells.

e. Telophase Karyokinesis is completed durin g telophase, and reor ganization of the contents of the twodaughter cells (cytokinesis) b egins. It is often difficult to distinguish late anaphase fro mearly telophase in the cells of plan ts. During telophase, however, a cell plate, the firstindication that cytokinesis is beginning, starts to form as a fine line across the center of th ecell. When co mplete, the cell plate divides the or iginal cell into two d aughter cells. Astelophase progresses, the nuclei begin to reor ganize: the chromosomes uncoil and becomelonger and thinner, the nuclear membrane reforms, and the nucleoli reappear. Mitosis endswith the assembly of two int erphase nuclei, each with one complete set of single-strandedchromosomes. The daughter cells resulting from mitotic division have the same number andkinds of chromosomes (and therefore the same genetic makeup) as the original cell.


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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 humanbody cell?

b)How many genes do scientists estimate ar e loc ated on thosechromosomes?

2.a)What do most genes contain instructions for building?

b)What functions do these perform?

3.How many pairs of genes are t hought to code for human skincolor?

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 humandiversity, 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 adisease caused by a dominant allele is_________________________.


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 anormal 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 usuallytreatable through ________________ and __________________.

19.Most of us carry _________ lethal recessive genes.


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.

mitosis, meiosis and chromosomes 171l/lab 08 mitosis... · mitosis, meiosis and chromosomes...

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