chapter  22
28 Pages

Localization of Gene Expression

In order to study cells, their structures and functions should be observed. The structure can be observed by using light and electron microscopy (see Chapter 21). The functions can be studied by obtaining fractionated organelles that are relatively pure. Each organelle has its own characteristics, such as size, shape, and density. These characteristics make one organelle different from other organelles within a cell. The procedures of fractionation consist of two consecutive stages, namely, (1) homogenization, which breaks cells open and releases organelles, and (2) centrifugation, which separates the individual organelles based on the organelles’ characteristics.1 Cells should be broken open gently, after which each of its organelles can be subsequently isolated. This homogenization is accomplished by physical breaking, ultrasonification, osmotic stress, and compression.2 Separation of organelles from one another is achieved by differential centrifugation. Centrifugal force is used to separate organelles as a function of their sedimentation coefficients, which are based on their size, shape, and density. Cells are homogenized and subjected to several steps of centrifugation. At each step, the pellet obtained contains an organelle fraction, and the supernatant is collected for the next step. For instance, the first step precipitates the nuclei, and the resulting supernatant contains the other cellular organelle fractions. At the second step, mitochondria, chlo-roplasts, and lysosomes are collected in the pellet, and the supernatant is used for the next step. Further fractionation of cell organelles, such as microsomes, is possible by the method of rate-zonal centrifugation.3 Sucrose or Percoll density gradients can be developed using the differential centrifugation methodology. In this case, the centrifugation medium is characterized by an increase in density. The reason why sucrose and Percoll are used for density gradients is due to their chemical natures. They are inert toward the centrifuge tubes, do not interfere with monitoring of the sample material, are easy to separate from each fraction after centrifugation, are easily used to observe the concentration of the gradient medium, are stable in solution, are available in a pure and analytical form, and exert minimal osmotic pressure. Organelle particles separate according to their own centrifugal field, size, shape, and density differences between the particles and the suspending medium. In order to analyze the relative purity of each

fraction, performance of activity assays of “marker enzymes” is recommended (see Table 22.1). A marker enzyme is an enzyme that is characteristic of each particular organelle in a given fraction.