Eukaryotic Cell Division Cells must continually grow and divide in order for an organism to grow, maintain its structure, and reproduce. Cell division involves the replication, or copying, of the complete set of hereditary information. It also involves the equal distribution of the genetic material in the resulting cells. The hereditary information of organisms is contained in large molecules called deoxyribonucleic acid, DNA. Objectives Upon completion of this laboratory you will be able to: 1. list the similarities and differences between mitosis and meiosis. 2. describe and recognize the stages of mitosis under a microscope or on models. 3. know the events that occur during each stage of mitosis and meiosis. 4. define the boldface terms. Chromosomes: Chromosomes (Figure 1) are tiny rod-shaped structures in the cell’s nucleus that carry the genetic message. A single complete set of chromosomes for an organism is referred to as the haploid number of chromosomes or 1n. Most organisms are diploid, having two complete sets of chromosomes, 2n. All species contain a specific number of chromosomes; for example humans have 23 pairs of chromosomes (for a total of 46). The matched chromosomes are called homologous chromosomes. They are alike in their size, structure, and the genes that they carry. Thousands of genes may be located on a single chromosome. A gene is the set of instructions for one protein product. It is estimated that humans have approximately 30,000 genes. Electron micrograph Artist’s rendition Chromosome under increasing magnification Figure 1. Several renditions of the structure of a chromosome. Spring 2006 1 Genetic material doesn’t always appear as it does in Figure 1. Usually it exists as loose strands of DNA and protein. A completely uncoiled human DNA strand in a single cell can be up to three feet long! Just before cell division the DNA strand coils, condensing 100X to form a chromosome. This structure helps prevent tangling and breakage of the genetic material during cell division. Two Cells From One – Mitosis Even after you are completely grown your somatic cells continue to grow and divide. Somatic cells are those cells that make up the structure of the body and include all the cells in the body except reproductive cells. Thus somatic cells are not involved in passing the genetic information from one generation to the next. A somatic cell’s nucleus divides through a process called mitosis. Following a mitotic cell division the two resulting daughter cells are genetically identical to each other and to the parent cell. The original parental cell and the two resulting daughter cells contain the same number of chromosomes and, barring mutations, possess identical copies of the same genetic blueprint. Procedure: 1. Watch the animations on mitosis: Plant cell mitosis: http://web.grcc.cc.mi.us/biosci/pictdata/mitosis/planmito.htm Animal cell mitosis: http://www.cellsalive.com/mitosis.htm 2. Obtain a prepared slide of Allium (onion) root tip. Root tips in plants contain cells that are constantly undergoing mitotic divisions. 3. Using the 430x objective select a region of the root tip that is one cell thick from the area designated in Figure 2; the mitotic stages will be clear here. Locate a region that contains the mitotic stages shown in Figure 3. 4. Work with your partner to quiz each other on your ability to identify the different mitotic stages. Spring 2006 2 Figure 2. Allium (onion) root tip. Interphase Prophase Metaphase Anaphase Telophase Figure 3. Stages of Plant Cell Mitosis 5. Draw in the labeled space provided the appropriate mitotic stage (prophase, metaphase, anaphase, and telophase) as it appears to you while observing it with the microscope. The Stages of Mitosis Interphase: The genetic material replicates, or copies, itself. Instead of having the usual 2 copies of genetic material the cell now has 4 (= 4n). The genetic material is dispersed throughout the nucleus. The nucleolus is usually visible, looking like a nucleus within the nucleus. The cell is now prepared to proceed with division. The pictures you draw for the following stages should resemble the one on the right. Be sure to label your drawings! Spring 2006 3 Prophase: The genetic material in the nucleus loops and coils forming visible thread-like structures called chromosomes. In animal cells the spindle apparatus forms and functions in separating the chromosomes during division. It consists of a star-like formation of fibers running from one end of the cell to the other. This structure is not visible in plant cells like the ones you are observing. The nuclear envelope and nucleolus start to disappear. Metaphase: Fibers from the fully formed spindle apparatus are attached to the constricted region in each chromosome called a centromere. The replicated chromosomes are lined up midway between the two ends of the cell. This imaginary midline is called the spindle equator. The nuclear envelope has completely disappeared. Anaphase: The replicated chromosomes begin to separate. The division of the centromeres and shortening of spindle fibers accomplish this. The centromeres divide separating the replicated chromosomes. As the two chromosomes divide each one goes to opposite ends of the cell. Note: The cell is now in the process of restoring its normal diploid (2n) state. Spring 2006 4 Telophase: The chromosomes are now clustered together at opposite ends of the cell. The chromosomes start to unwind and may no longer be clearly visible. The spindle apparatus disappears and the nuclear envelope reforms. Cytokinesis: Division of the cytoplasm usually follows nuclear division. In plants the onset of cytokinesis is marked by the formation of a cell plate between the two nuclei in late telophase. In animals cleavage occurs when the cell membrane pinches in, or furrows, between the two telophase nuclei and starts to separate the cytoplasm. It may be difficult to differentiate between late telophase and cytokinesis. Time spent in mitotic stages. Procedure: Now you are going to calculate how long a specific stage of mitosis lasts in comparison to the other stages. This exercise assumes that the more cells there are in a specific stage, the longer that stage lasts. A nucleus undergoing division is called a mitotic figure. For this exercise find an area on the slide which has as many mitotic figures as possible. 1. At 430x count the total number of cells in one field of view. Include all cells, both mitotic and non-mitotic. Record this number in Table 1 next to “Total # of Cells”. 2. Count the number of cells in prophase and record in the appropriate column in Table1. Spring 2006 5 3. Do the same with metaphase, anaphase, and telophase nuclei. 4. Add together the number of cells undergoing mitotic division: (# in prophase) + (# metaphase) + (# anaphase) + (# telophase) = (total # of mitotic cells) Total # of mitotic cells = _______________________ 5. Total number of cells – total mitotic cells = _____cells in interphase. Record this in the appropriate column in Table 1. 6. With the following equation, calculate the duration of each mitotic stage using the data from the entire class: duration of mitotic stage (in hours) = number of cells in a stage x 24 hr. total number of cells Table 1. Duration of mitotic stages in onion root tip cells. # of cells you counted Total number of cells Class total # of cells Duration of stage (hrs.) …………… Number in prophase Number in metaphase Number in anaphase Number in telophase Number in interphase Two Halves Make a Whole: Meiosis In sexually reproducing organisms, egg and sperm cells combine their genetic information to produce a new, unique organism. Gametes, or sex cells, are different from other body cells because each contains only one copy of the genetic blueprint instead of the usual two. In other words, they are haploid (1n). If gametes contained the usual two sets of chromosomes, each new generation would have twice the number of chromosomes. Just imagine! If this really happened, the 46 chromosomes in your cells would be increased to 368 in your great-grandchildren and 924 chromosomes in their great-grandchildren! Meiosis is the process of cell division that prevents this wild escalation in the number of chromosomes contained in our cells. Remember that meiosis occurs only in the germ cells, the reproductive cells that carry the genetic information from one generation to the next. In female animals meiotic cell division results in one functional egg and three Spring 2006 6 nonfunctional cells called polar bodies. In males each full meiotic division produces four functional sperm. Refer to your textbook for help in the study of the stages of meiosis. Important Differences Between Meiosis and Mitosis: 1. In prophase I of meiosis there is a pairing of homologous chromosomes called synapsis. These chromosomes have already replicated, so four chromosomes are joined together forming a tetrad. This does not occur during mitosis. 2. In prophase of meiosis crossing-over occurs. At this time the matched homologous chromosomes break at exactly the same places and exchange similar segments. This process effectively exchanges similar genes between the matched and paired chromosomes. Genes come in alternative forms called alleles. For example the gene for eye color may have several alternative forms (blue or brown). There is no gain or loss of genetic material during crossing-over but rather an exchange of the same genes, (but sometimes different alleles), one for one. Thus, new genetic combinations on a chromosome may be formed. This is recombination. 3. The cells in meiosis go through a second successive division without first doubling their genetic material. This result in four cells called gametes. Each of these cells contains only one copy (1n) of the hereditary blueprint for the organism. Meiosis-demonstrating the process. Procedure: As directed by your instructor, demonstrate meiosis using the following materials: 4 chromosomes (chains of pop beads) 2 centrioles 4 centromeres 2 nuclear envelopes spindle fibers The beads on two chains should be yellow while those on the other two chains red. A magnet would be placed in the center of each chain. These chains of beads represent homologous chromosomes (one contributed by Mom and one contributed by Dad) with each bead being a gene and the magnet being the centromere. Mark a “gene” as shown by your instructor at the same site (locus) on each strand as shown in Figure 4. Spring 2006 7 Figure 4. Pop bead chromosomes with marked genes Interphase: The “chromosomes” you obtained have already replicated and are in the interphase stage. The two sets of replicated chromosomes are dispersed in the nucleus and the nuclear envelope is present as represented by the string. In animal cells centrioles, from which the spindle apparatus will arise, are near the nucleus and at right angles to each other. Prophase I: The two homologous chromosomes pair with each other. This is called synapsis. To simulate synapsis slide the homologues together and entwine them (two like strands entwined with the other two like strands). See Figure 5A. Crossing-over, the exchange of matched genetic material, occurs now. For crossing-over detach a small section in one strand of each replicated chromosome and exchange them. See Figure 5B. The nuclear envelope is still present, but it is beginning to disappear. 5A. Synapsis 5B. Crossing-over Figure 5. Synapsis and crossing-over of chromosomes during Prophase. Spring 2006 8 Metaphase I: Line the chromosomes up at the imaginary equator of the cell. The centromeres of each homologue lie on the equator. Move the centrioles so that they are at opposite ends of the cell (see Figure 6). Figure 6. Chromosomes at metaphase. Anaphase I: The homologous chromosomes separate with each homologue moving to opposite poles. Note that each homologue is still in its replicated condition. Separate the homologues. (Figure 7.) Figure 7. Separation of homologous chromosomes during Anaphase I. Telophase I: Pile the chromosomes up near their respective poles. A new nuclear envelope will surround each set of chromosomes. Furrowing of the membrane occurs to form two new daughter cells. There are now two daughter cells with a cell membrane between the two new nuclei. Draw your pop bead chromosomes as they appear after meiosis I in the two nuclei labeled “after meiosis I” Spring 2006 9 Daughter Cell #1 after Meiosis I Daughter Cell #2 after Meiosis I Interkinesis: The first meiotic division is now complete and two cells are formed. Cytokinesis has occurred and the nuclear envelope has reformed. It is important to note that DNA replication does not occur prior to the second division. The spindle reforms to provide a complete spindle apparatus for each daughter cell. Meiosis II Prophase II: The replicated chromosomes are still attached by their centromeres. During prophase II the nuclear envelope disorganizes and the chromosomes recondense. Remember this is occurring in two cells simultaneously. Metaphase II: Align the replicated chromosomes at the equator of the cell. Anaphase II: Separate the replicated homologues and their centromeres. The individual daughter chromosomes should now be heading towards opposite poles of the cell. Telophase II: Pile up the daughter chromosomes once they are at opposite poles of the cell. The nuclear envelope reforms and the cytoplasm divides. Four daughter nuclei now exist. Each nucleus contains one individual chromosome, one-half the number present in the original cell. Draw your pop bead chromosomes as they appear after meiosis II in the space provided labeled “Gamete nuclei”. (Don’t forget to include the crossover that occurred in Meiosis I.) Spring 2006 10 Four new daughter cells after meiosis II Gametogenesis: In this meiosis exercise you simulated spermatogenesis, the production of four sperm cells from one parent cell. The female process of oogenesis (see below) is somewhat different. Due to unequal division of cytoplasm, one functional egg and three “runts” called polar bodies are formed. Polar bodies are almost always non-functional. Fill in Table 2, Mitosis vs. Meiosis Table 2. Mitosis vs. Meiosis: a comparison of characteristics. Mitosis Meiosis Number of divisions Number of daughter cells Chromosome number in daughter cells (haploid or diploid?) Daughter cells identical to each other and parent cell? (yes or no) Produces gametes (yes or no)? Spring 2006 11