Gene Control

David Latchman

• Editore: Garland Science
• Anno: 2020
• ISBN:
  9780429637599
• Edizione: 2
• Argomento: Ciencias biológicas
• Categoria: Biología

The new edition of Gene Control has been updated to include significant advances in the roles of the epigenome and regulatory RNAs in gene regulation. The chapter structure remains the same: the first part consists of pairs of chapters that explain the mechanisms involved and how they regulate gene expression, and the second part deals with specific biological processes (including diseases) and how they are controlled by genes. Coverage of methodology has been strengthened by the inclusion more explanation and diagrams.

The significant revision and updating will allow Gene Control to continue to be of value to students, scientists and clinicians interested in the topic of gene control.

Levels of Gene Control 1

Introduction

The evidence that eukaryotic gene expression must be a highly regulated process is available to anyone visiting a butcher’s shop. The various parts of the mammalian body on display differ dramatically in appearance, ranging from the muscular legs and hind quarters to the soft tissues of the kidneys and liver. However, all these diverse types of tissues arose from a single cell, the fertilized egg or zygote, raising the question of how this diversity is achieved.

It is clear that regulatory processes must exist that allow different cells to form in different places in an orderly manner during embryonic development and to maintain their differences in the adult organism. Moreover, cells need to be able to alter their pattern of gene expression in response to changes such as alterations in the levels of nutrients, growth factors, hormones, etc.. It is the aim of this book to consider the processes regulating tissue-specific gene expression in eukaryotes and the manner in which they produce these differences in the nature and function of different tissues and cell types.

1.1 The Protein Content of Different Cell Types is Different

The fundamental dogma of molecular biology is that DNA produces RNA which in turn produces proteins. The genetic information in the DNA specifying particular functions is converted into an RNA copy which is then translated into protein. The action of the protein then produces the phenotype, be it the presence of a functional globin protein transporting oxygen in the blood or the activity of a proteinaceous enzyme capable of producing the pigment causing the appearance of brown rather than blue eyes. If the differences in the appearance of tissues described above are indeed caused by differences in gene expression in different tissues, they should be produced by differences in the proteins present in these tissues and the cell types of which they are composed. Such differences can be detected both by specific methods that study the expression of one particular protein and by general methods aimed at studying the expression of all proteins in a given tissue.

Specific methods can be used to study the expression of individual proteins in tissues and cells

A simple means of determining whether differences in protein composition exist in different tissues or cell types involves investigating the expression of individual known proteins in such tissues using a specific antibody to that protein. Such antibodies can be used in conjunction with the one-dimensional polyacrylamide gel electrophoresis technique to investigate the expression of a particular protein in different tissues. In this technique, proteins are denatured by treatment with the detergent sodium dodecyl sulfate (SDS) and then subjected to electrophoresis in a polyacrylamide gel, which separates them according to their size.

Subsequently, in a technique known as Western blotting, the gel-separated proteins are transferred to a nitrocellulose filter which is incubated with the antibody. The antibody recognizes and binds specifically to the protein against which it is directed. This protein will be present at a particular position on the filter, dependent on how far it moved in the electrophoresis step and hence on its size. The binding of the antibody is then visualized by an enzymatic or fluorescent detection procedure. If a tissue contains the protein of interest, a band will be observed in the track containing total protein from that tissue and the intensity of the band observed will provide a measure of the amount of protein present in the tissue. If none of the particular protein is present in a given tissue, no band will form (Figure 1.1).

FIGURE 1.1
Western blot with an antibody to guinea pig casein kinase showing the presence of the protein in lactating mammary gland (A) but not in liver (B). Courtesy of Alison Moore, from Moore A, Boulton AP, Heid HW et al. (1985) Eur J Biochem 152:729–737. With permission from Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc.

This method allows the presence or absence of a specific protein in a particular tissue to be assessed using one-dimensional gel electrophoresis without the complicating effect of other unrelated proteins of similar size, since these will fail to bind the antibody. Similarly quantitative differences in the expression of a particular protein in different tissues can be detected on the basis of differences in the intensity of the band obtained when extracts prepared from the different tissues are used in this procedure.

The results of such experiments indicate that while some proteins are present at similar abundance in all tissues, the abundance of other proteins varies between different tissues and a large number are specific to one or a few tissues or cell types. Thus, the differences between different tissues in appearance and function are correlated with qualitative and quantitative differences in protein composition, as assayed using antibodies to individual proteins.

General methods can be used for studying the overall protein composition of tissues and cells

As well as examining the expression of individual proteins in different tissues, it is also possible to use more general methods to compare the overall protein composition of different tissues. All the proteins present in a tissue can be subjected to one-dimensional gel electrophoresis and examined by using a stain that reacts with all proteins. This method allows the visualization of all cellular proteins separated according to their size on the gel, rather than using Western blotting with a particular antibody to focus on a specific protein. However, this method is of relatively limited use to investigate variations in the overall protein content of different tissues. This is because of the very large number of proteins in the cell and the limited resolution of the technique, which separates proteins solely on the basis of their size. For example, two entirely different proteins in two tissues may be scored as being the same protein simply on the basis of a similarity in size.

A more detailed investigation of the protein composition of different tissues can be achieved by two-dimensional gel electrophoresis. In this procedure (Figure 1.2), proteins are first separated on the basis of differences in their charge, in a technique known as isoelectric focusing. The separated proteins, still in the first gel, are subsequently layered on top of an SDS-polyacrylamide gel and separated by electrophoresis according to their size. Thus, a protein moves to a position determined by both its size and its charge. The much greater resolution of this method allows a number of differences in the protein composition of particular tissues to be identified. Some spots or proteins are found in only one or a few tissues and not in m...