Lab # 2: Mitosis and the Cell Cycle 2012_2013 Practical Of Genetics BIOL 2121 Lab # 2: Mitosis and the Cell Cycle 2012_2013

Objectives: To prepare slides of onion root tips demonstrating the stages of mitosis (somatic cell division) and identification of cells in the various stages of mitosis.

Background: Prokaryotic cells (bacteria) reproduce asexually by binary fission. Bacterial cells have a single circular chromosome, which is not enclosed by a nuclear envelope. During binary fission the bacterial chromosome is duplicated, the cell elongates, and the two chromosomes migrate to opposite ends of the cell. Each daughter cell receives one chromosome and is identical to the parent cell. Binary fission is a relatively fast and simple process.

Cont. Eukaryotes are diploid, which means they have two sets of chromosomes; one set of chromosomes is inherited from each parent. For example in a growing plant root, the cells at the tip of the root are constantly dividing to allow the root to grow. Because each cell divides independently of the others, a root tip contains cells at different stages of the cell cycle. This makes a root tip an excellent tissue to study the stages of cell division.

Cont. The cell division cycle : is a sequence of events in a eukaryotic cell between one mitotic division and another. It includes : 1. Interphase (G1 phase, S phase, and G2 phase) 2. M phase. In the M-phase both the nucleus and the cytoplasm divide (mitosis and cytokinesis). Chromosomes are visible only during mitosis when they separate between the daughter cells. The process of mitotic division ensures that both daughter cells will gain identical set of chromosomes containing identical genetic information.

Cell cycle: G1 - initial growth cycle S - DNA synthesis (replication) G2 - second growth stage M - Mitosis and cytokinesis. G1+S+G2= Interphase

Duration of the cell cycle in Human cells (in hours) It takes about 16 hours Interphase Mitosis G1 S G2 M 5 7 3 1 Duration of phases of Mitosis: Prophase: 36 minutes Metaphase: 3 minutes Anaphase: 3 minutes Telophase: 18 minutes Vary among cell types Consistent among cell types

Interphase Chromatin appears dispersed, DNA replication occurs. (Chromatin is a mass of uncoiled DNA and associated proteins called histones).

Mitosis Mitosis, or nucleus division, is the first part of M-phase and in consists of four stages (prophase, metaphase, anaphase and telophase).

Mitosis is the first part of M-phase and divided into four distinct phases: 1. Prophase: Chromatin condenses chromosomes become visible nuclear envelope and nucleoli disappear spindle starts to form attach to the kinetochore ( a portion of the centromere). Each replicated chromosome comprises two chromatids, both with the same genetic information (called sister chromatids).

Cont. Prometaphase: In this stage the nuclear envelope breaks down so there is no longer a recognizable nucleus. Some mitotic spindle fibers (microtubules) elongate from the centrosomes and attach to kinetochores (protein bundles at the centromere region on the chromosomes where sister chromatids are joined).

Cont. These microtubules which attach to the kinetochore are called “kinetochore microtubules”. Other spindle fibers elongate but instead of attaching to kinetochores, they attach with spindle fibers growing from the other side of the cell. These are called “polar microtubules or non-kinetochore microtubules”. The chromosomes start to migrate towards the center of the cell by the kinetochore microtubules

2. Metaphase All chromosomes align in one plane at the center of the cell called the equatorial plane (also referred to as the metaphase plate).

Anaphase: Spindle fibers shorten the kinetochores separate the sister chromatids (daughter chromosomes) are pulled apart and begin moving to the cell poles.

Telophase: Telophase, the last stage of division, is marked by a noticeable condensation of the chromosomes, followed by formation of a new nuclear envelope around each group of chromosomes. The chromosomes gradually de-condense to form the chromatin seen in interphase. The nucleoli reappear.

Cytokinesis ( cytoplasmic division) Usually occurs at the end of telophase. In plant cells cytokinesis is accomplished by the formation of a cell plate. Animal cells separate by forming a cleavage furrow.

Preparing An Onion Root Tip Squash: It is possible for you to make your own stained preparations of onion root tips and observe mitotic figures (2n=16). Onion bulbs have been rooted in water. Growth of new roots is due to the production and elongation of new cells. Mitotic divisions are usually confined to the cells near the tip of the root. Follow the procedure outlined below to make your own root tip preparation.

Root of Onion

Why use onion roots for viewing mitosis? The roots are easy to grow in large numbers. The cells at the tip of the roots are actively dividing, and thus many cells will be in stages of mitosis. The tips can be prepared in a way that allows them to be flattened on microscopes slide (“squashed”) so that the chromosomes of individual cells can be observed. The chromosomes can be stained to make them more easily.

Materials: Onion bulb with roots Crystal violet and other stains. Fixative solution (1 part glacial acetic acid to 3 parts ethanol). 1M HCl Clean slide & cover Forceps Razor blade Fingernail polish

Procedure: 1. Obtain two small cups, label one “ HCL” and the other “Carnoy.” Pour in enough of each to just cover the bottom of its cup. 2. Obtain an onion bulb that is just beginning to show the emergence of roots. Cut a small piece of root off and use forceps to place it in the cup of 1M HCL for 4 min at 60ºC.

3. Transfer the root into the cup of carnoy fixative 3. Transfer the root into the cup of carnoy fixative. The root should remain in the carnoy fixative for 4 minutes. 4. After the 4 minutes, place the root on a slide. Then, cut off the bottom 1 or 2mm of the root tip & discard the rest. 5. Cover the root with a 1drop of Crystal Violet for 2 minutes. After the 2 minutes, blot away the stain; be careful not to touch the root tip.

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6. When the stain has been blotted away, cover the root tips with drops of water to remove the stain. Apply water drops until water runs mostly clear. Blot slide without touching root tip. 7. Gently lower a cover slip over the root tip. Cover the slide with a paper towel and with your thumb (or a pencil eraser), firmly press on the cover slip; Do not twist the cover slip. The pressure will spread the cells into a single layer.

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Cont. 8. Then using the fingernail polish, carefully paint the edges of the cover slip. Make sure not to move the cover slip. Finally allow the slide to dry. 9. Now you are ready to view you slide under low power. Place your prepared slide on the microscope. 10. Use the low power objective on your microscope to look for thin layers of cells and then use the 40X power objective to observe mitotic stages in individual cells.

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With Best Wishes