Laboratory 3 DNA, Chromosomes and Mitosis

Transcription

1 Laboratory 3 DNA, Chromosomes and Mitosis Purpose In this lab you will extract chromosomal DNA from cow liver and identify onion root cells in different stages of mitosis. Introduction Part I. DNA and Chromosomes DNA is the carrier of genetic information for most of the living organisms (some are carried by RNA). A DNA molecule consists of two long polynucleotide chains. Each of these chains is composed of four types of nucleotides. DNA encodes genetic information in the sequence of the nucleotides along each chain. In eukaryotic organisms, the DNA in the nucleus is distributed among a set of different chromosomes. Each chromosome consists of a single enormously long linear DNA molecule associated with proteins that fold and organize the DNA into a more compact structure called chromatin. Although chromatins are one thousand times more compact than the DNA molecules themselves, chromatins in a non-dividing cell are long, thin, tangled threads and cannot be easily distinguished under the light microscope. In an actively dividing cell however, the chromatins condense further and take on a distinct fourwinged shape that is visible under a light microscope. Part II. Mitosis Cells reproduce by duplication their contents and dividing in two. The cell cycle has two phases: interphase and M phase. Interphase is a time of cell growth and DNA replication. Most cells spend the majority of time in this stage. Interphase contains three sub-phases: G1, S, and G2. G1 (or Gap 1) This phase is variable in length. If a cell is not going to divide, it may remain here until it dies. Cells that will divide increase in size, produce RNA, and synthesize protein in preparation for the next stage. S Phase (Synthesis) The cell s genetic material is replicated through DNA synthesis. G2 (or Gap 2) The cell continues to grow and prepare for mitosis. After interphase, mitosis begins. Mitosis has four main stages: prophase, metaphase, anaphase, and telophase. Prophase In this stage, the centrioles move to opposite poles of the cell and begin to form an array of microtubles called the spindle. The chromosomes become more compact. The surface 1

2 that encloses the nucleus, known as the nuclear envelope, breaks into small vesicles, allowing the spindle into the nuclear region. Each sister chromatid attaches to a microtuble originating from opposite poles, which results in the movement of chromosomes. Metaphase Gradually, the chromosomes move to the middle region between each pole called the equatorial plate (metaphase plate). The cell is in metaphase when all centromeres are at the equatorial plate. The chromosomes are at their most compact at this time. Anaphase The centromeres of each sister chromatid separate, forming daughter chromosomes. Daughter chromosomes move to opposite poles of the spindle. Telophase When the chromosomes have stopped moving, the nuclear envelope reforms around each cluster. Chromosomes uncoil and become the tangle of chromatin, characteristic of interphase. The last phase in the cell cycle is cytokinesis. Cytokinesis is the division of the cytoplasm. It begins in anaphase but is not completed until after the two daughter nuclei have formed. In animal cells, a cleavage furrow forms at the equator and eventually pinches the parent cell in to two. In plant cells, membrane bound vesicles migrate to the equator and fuse to form the cell plate. This eventually extends across the cell and divides the cytoplasm into two. Following cytokinesis, both daughter cells contain all the components of a complete cell. Procedures Part I. Extract DNA from cow liver 1. Place a small chunk of liver (about 4 g) into a mortar. Mince the liver into small pieces with scissors for about one minute. 2. Add 10 ml 0.9% NaCl to the well-diced liver. Grind the liver thoroughly (for about 5 minutes) with the pestle using a circular motion. 3. Strain the liver cell suspension through several layers of cheesecloth into a 50 ml beaker to remove any remaining chunks. 4. Add 3 ml 10% SDS and swirl the beaker gently. The SDS will break the liver cell membrane and the nuclear membrane. You will see the turbid cell suspension becoming a clear cell lysate. The cell lysate is a mixture of all the cell components in its native forms, including proteins and DNAs. 5. Tilt the beaker and slowly add 20 ml 95% ethanol along the side of the beaker. At the beginning you will see white string like structures develop at the interface between ethanol and the cell 2

3 lysate. This is DNA strands coming out of solution. As more ethanol is added, more DNA strands will come out of the solution and form a matt like substance. 6. Using a glass stirring rod, gently and slowly stir the mixture. The DNA will collect slowly onto the rod. Pick up the rod slowly, if you are lucky, you might see an unbroken strand between the rod and the mixture. Part II. Mitosis: Observation of cell cycle stages using onion root In an onion, the root cap protects the root tip in the soil. Just above the root cap is the zone of cell division. As cells divide, the root is pushed further in the soil. The mature cells elongate and differentiate into specialized cells. Because of the rapid cell division, the onion root is a good place to look for the stages of mitosis. 1. Add I ml 1N HCl in a micro-centrifuge tube. Caution: HCl is a strong acid. Wear gloves when handling it and immediately rinse it off if some get on your skin. 2. Using scissors, cut 2 roots tips about 1 cm long, and transfer them into the tube containing HCl. 3. Place the tube in a 60 O C water bath, and allow the roots to incubate for 12 minutes. 4. Using forceps carefully transfer one root tip to the center of a clean microscope slide. 5. Using a razor blade cut off most of the root leaving ~2mm of the tip. 6. Cover the root tip with a drop of the Aceto-orcein stain. Caution: Aceto-orcein stain contains strong acid. Wear gloves when handling it and immediately rinse it off if some get on your skin. 7. Incubate the root in the stain for 2 minutes. 8. Rinse the root tip carefully with di water over a beaker. Do not wash the root tip off the slide. 9. Cover the root tip with a cover slip, and then carefully push down on the cover slide with your thumb or a pencil. Push hard, but do not twist or push the cover slide sideways. The root tip should spread out to a diameter about cm. 10. Mount the slide onto a compound microscope and scan the onion root under the 10x objective. Look for the region that has large nuclei relative to the size of the cell; among these cells will be found cells displaying stages of mitosis. 11. Switch to the 40X objective to make closer observations. Locate each stage of the cell cycle, Draw a cell in the designated boxes in the post-lab assignment sheet. Label the nucleus, chromatin and mitotic spindle when appropriate. Part III. Mitosis: Estimation of time spent in each phase You can approximate how much relative time is spent in each phase of the cell cycle by making snapshot observations of a stained onion root. By counting up the number of cells observed in each phase, you can determine the proportion of cells in each phase. This estimates the proportion of time spent in each phase. Since prophase and pro-metaphase are difficult to distinguish, classify all these cells as prophase. You can switch back to the 10X objective and track the cells you counted by counting in rows. Every group should count ~50 random cells (or all the cells in view under 10X). Record your data below and then add to the class spreadsheet on the computer. 3

4 Phase Number of Cells Class Totals Interphase Proportion of cells (number divided by total) Prophase Metaphase Anaphase Telophase Total 1.00 (or 100%) 4

5 Name: BU ID: Teaching Fellow name and section number: Laboratory 3 DNA, Chromosomes and Mitosis Pre-lab Questions 1. What molecule carries the genetic information of a living organism and where are these molecules located in a eukaryotic cell? (20 pts) 2. Besides DNA, what is the other major component of a chromosome and what is its function? (20 pts) 3. What happens during the interphase of the cell cycle? (20 pts) 4. What are the four stages of mitosis? (20 pts) 5. Why can t you see the chromosome during interphase of the cell cycle? (20 pts) 5

6 Laboratory 3 DNA, Chromosomes and Mitosis Post-lab Assignment 1. Draw an onion root cell in each stage of the cell cycle. Label the nucleus, chromatin and mitotic spindle when appropriate. (50 pts) Interphase Prophase Metaphase Anaphase Telophase 2. Based upon the class results, order the stages of mitosis from shortest to longest. Why do you think the shortest being the shortest and the longest being the longest based on what happens in the cell in each stage? (50 pts) 6