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The remarkable diversity of form and function that eukaryotic cells assume is even more remarkable when you consider that a multicellular organism begins life as a single cell, the zygote. The egg and sperm (the gametes), though usually unequal in size, give an equal number of chromosomes to the zygote: each contributes the haploid number of chromosomes. The developing organism, therefore, contains a diploid number of chromosomes – a haploid set from each parent. For example, the diploid complement of a human zygote (and subsequently all other body cells) is 46 chromosomes; the haploid complement of each gamete is 23 chromosomes. After fertilization, the zygote gives rise to all the cells that make up the organism by repeated cell divisions, called mitosis. In multicellular organisms, mitosis permits growth and repair of tissues. In eukaryotic unicellular organisms, mitosis is a form of asexual reproduction.
During the formation of the eggs and sperm, the chromosome number is reduced from diploid to haploid by a type of cell division called meiosis. We will examine this form of cell division later in this laboratory exercise.
THE CELL CYCLE:
The complex series of events that encompasses the life span of an actively dividing cell is termed the cell cycle, which includes an interphase during which copy of the DNA) occurs, as well as the synthesis of RNA and proteins. Note that replication of DNA occurs only during a period of interphase called the S phase (S for “synthesis”). The doubling of DNA during the S phase provides a full complement of DNA for the 2 daughter cells that will result from the ensuing mitotic division. During interphase, there are also 2 phases called G1 and G2. (G for “gap”). The G1 phase, which precedes DNA replication, includes a variety of growth processes and synthesis of compounds other than DNA. During the G2 phase, which immediately follows DNA replication, the molecules. and structures directly involved with mitosis are synthesized, assembled. The combination of G1, S, G2, and M phases make up the cell cycle (mitotic cycle).
For the convenience of scientists, the process of mitosis is divided into 4 distinct stages: prophase, metaphase, anaphase, and telophase. The mitotic process usually occupies less than 10% of the total time taken by the cell cycle. Thus, mitosis is only part of the overall cell cycle.
Mitosis is essentially the same in all organisms but, just as plant and animal cells differ to some extent structurally, there are some differences between them in this process. It is the objective of this part of the lab exercise to examine the essential steps in mitosis and to characterize the similarities and differences in this process between plant and animal cells.
1. MITOSIS IN PLANTS CELLS
The onion (Allium) root tip is one of the most widely used materials for the study of mitosis because it is readily available, preparation of the dividing cells is easy, and the chromosomes are large and few in number. Root tips of plants contain meristems, which are localized areas of rapid cell division; therefore, chances are good that in a specimen of such tissues, one can find every stage of mitosis.
Obtain a slide of onion root tips, and note a series of dark streaks on it. Each streak is a very thin longitudinal section through an onion root tip.
Place the slide on the stage of your compound microscope, and locate 1 of the sections under low power ( I0X). It is often possible to determine whether a given section shows good examples of mitotic stages. Because each section is very thin, not all will be equally good for study. After your preliminary examination under low power, change to high power (40X or 43X), being careful not to break the slide. Keep in mind the sequence in which the stages occur, but do not try to find them in sequence. Thus, if you happen to find an anaphase first, study it before proceeding to another stage. Because cells remain in interphase and prophase longer than in the other stages, chances are that most of the cells will be in interphase; many will be in prophase; and only a few will be in the other stages.
The interphase cell, so named because early biologists thought it was a resting phase, is actively undergoing cellular respiration and the synthesis of DNA, RNA, and protein. Interphase cells are characterized by a distinct nucleus bounded by a nuclear envelope. One or more nucleoli may be visible.
During prophase, the DNA has condensed enough for the chromosomes to become distinguishable in the nucleus. The nuclear envelope begins to break down, and the chromosomes are then distributed throughout the cytoplasm. As the chromosomes continue to compact, the nucleolus disappears. During prophase in the onion root tip, the chromosomes often appear as a coiled mass. Even at this early stage, each chromosome has doubled, although this will be difficult to see on the slide. Under very high magnification, it is possible to see that each chromosome is composed of 2 separate strands, the sister chromatids. The 2 sister chromatids are identical. The sister chromatids are joined together at a region of attachment called the centromere. Within this region, each chromatid contains a disc-shaped kinetochore. A bridge of microtubules called the spindle apparatus begins to form, extending between the 2 poles of the cell. The spindle fibers insert into the kinetochores and run from them outward to the 2 poles of the cell.
Near the central region of the spindle apparatus is a plane referred to as the metaphase plate, at right angles to the long axis of the spindle fibers. During metaphase, the short, thick, double-stranded chromatids become arranged along the metaphase plate; the chromosomes are maneuvered into position by the spindle fibers that are attached to the kinetochores of each chromosome. One sister chromatid of each chromosome is connected to spindle fibers running to 1 pole, and the other sister chromatid is connected to spindle fibers running to the other pole. By the time the chromosomes are organized along the metaphase plate, the nuclear envelope has completely disintegrated.
Anaphase begins when the sister chromatids are pulled apart by the spindle fibers; the centromere splits, and the sister chromatids become daughter chromosomes as they are pulled toward opposite poles of the cell. This stage can be recognized in the onion cell by the 2 groups of V-shaped chromosomes; the sharp end of the V is oriented toward the pole. The onion has a diploid number of 16 chromosomes, so it is seldom possible to see all of them at 1 time.
Reduce the light by adjusting the diaphragm of your microscope, and see if you can find any spindle fibers near the center of the cell. They appear as very fine lines between the 2 groups of chromosomes, but they are often not visible in a study of this kind. Anaphase ends when the newly-separated daughter chromosomes arrive at opposite poles of the cell.
.It is often difficult to distinguish late anaphase from early telophase in the cells of the onion root tip. As telophase progresses, the nuclei begin to reorganize, the chromosomes uncoil and become longer and thinner, the nuclear envelope reforms by fusion of parts of the endoplasmic reticulum, and the nucleoli reappear. Mitosis ends with the assembly of 2 interphase nuclei, each with 1 complete set of single-stranded chromosomes.
Mitosis is completed during cytokinesis. The first indication that cytokinesis is beginning, a cell plate.starts to form as a fine line across the center of the cell. When complete the cell plate divides the original cell into 2 daughter cells. In some cells, the cell plate is indistinct. The daughter cells resulting from mitotic division have the same number and kinds of chromosomes as the original cell. Thus, in the onion, each daughter cell has 16 chromosomes.
2. MITOSIS IN ANIMAL CELLS
Mitosis in animal cells is easily observed on a prepared slide of a whitefish blastula. (A blastula is an early stage of development of an embryo formed by successive mitotic divisions.)
As in plant cells, animal cells in interphase are characterized by a distinct nucleus bounded by a nuclear envelope. The nucleolus should be identifiable. Immediately adjacent to the nuclear envelope is a cytoplasmic organelle known as the centrosome.
As in plant cells, mitosis begins with prophase. During prophase, in contrast to plant cells, 2 pairs of structures called centrioles form within the centrosome. They begin to move apart, migrating around the nucleus toward the opposite poles of the cell. Microtubules radiate from each pair of centrioles like spokes on a wheel, forming a configuration known as an aster. The centrioles continue to migrate until they lie at opposite poles of the cell. When the nuclear envelope disintegrates, the spindle becomes visible between the centrioles. Locate various stages of prophase on the slide.
During metaphase, the chromosomes move toward the central region of the spindle to form the metaphase plate. By the time the chromosomes are organized along the metaphase plate, the nuclear envelope has completely disintegrated. Locate this phase of mitosis on your slide.
The sister chromatids that make up each chromosome are now separated from each other and are pulled by the spindle fibers to opposite poles of the cell. The separated sister chromatids now become daughter chromosomes. When the daughter chromosomes reach the poles, anaphase ends and telophase begins.
During telophase, the spindle disappears, 2 daughter nuclei are organized, the nucleoli reappear, and the nuclear envelopes are reformed.
Cytokinesis, a deep cleavage furrow appears, as the cytoplasm becomes pinched in between the 2 nuclei, and cytokinesis takes place. This results in 2 daughter cells having equivalent nuclear components and equal amounts of cytoplasm.