The AGT Cytogenetics Laboratory Manual

Marilyn S. Arsham, Margaret J. Barch, Helen J. Lawce, Marilyn S. Arsham, Margaret J. Barch, Helen J. Lawce

• Publisher: Wiley-Blackwell
• Year: 2017
• ISBN:
  9781119061281
• Edition: 4
• Topic: Scienze biologiche
• Subtopic: Genetica e genomica

Cytogenetics is the study of chromosome morphology, structure, pathology, function, and behavior. The field has evolved to embrace molecular cytogenetic changes, now termed cytogenomics.
Cytogeneticists utilize an assortment of procedures to investigate the full complement of chromosomes and/or a targeted region within a specific chromosome in metaphase or interphase. Tools include routine analysis of G-banded chromosomes, specialized stains that address specific chromosomal structures, and molecular probes, such as fluorescence in situ hybridization (FISH) and chromosome microarray analysis, which employ a variety of methods to highlight a region as small as a single, specific genetic sequence under investigation.

The AGT Cytogenetics Laboratory Manual, Fourth Edition offers a comprehensive description of the diagnostic tests offered by the clinical laboratory and explains the science behind them. One of the most valuable assets is its rich compilation of laboratory-tested protocols currently being used in leading laboratories, along with practical advice for nearly every area of interest to cytogeneticists. In addition to covering essential topics that have been the backbone of cytogenetics for over 60 years, such as the basic components of a cell, use of a microscope, human tissue processing for cytogenetic analysis (prenatal, constitutional, and neoplastic), laboratory safety, and the mechanisms behind chromosome rearrangement and aneuploidy, this edition introduces new and expanded chapters by experts in the field. Some of these new topics include a unique collection of chromosome heteromorphisms; clinical examples of genomic imprinting; an example-driven overview of chromosomal microarray; mathematics specifically geared for the cytogeneticist; usage of ISCN's cytogenetic language to describe chromosome changes; tips for laboratory management; examples of laboratory information systems; a collection of internet and library resources; and a special chapter on animal chromosomes for the research and zoo cytogeneticist. The range of topics is thus broad yet comprehensive, offering the student a resource that teaches the procedures performed in the cytogenetics laboratory environment, and the laboratory professional with a peer-reviewed reference that explores the basis of each of these procedures. This makes it a useful resource for researchers, clinicians, and lab professionals, as well as students in a university or medical school setting.

CHAPTER 1
The cell and cell division

Margaret J. Barch¹ and Helen J. Lawce²

¹ *(deceased) formerly, Frank F Yen Cytogenetics Laboratory, Weisskopf Child Evaluation Center, University of Louisville, Louisville, KY, USA

² Oregon Health & Science University Knight Diagnostic Laboratory, Portland, OR, USA

1.1 The cell [1,2]

The cell is the basic unit of life – the simplest structure capable of independent existence. The simplest organisms consist of only one cell. Higher organisms are composed of complex colonies of interdependent cells, each colony with a specialized function necessary for the survival of the organism. Cells that have the same general function are often grouped together to form tissues, such as muscle, bone, and connective tissue. Tissues may be combined in larger functional units called organs, such as kidneys, skin, and heart. Organs can in turn be grouped by function into organ systems, such as the respiratory and circulatory systems.

Cells vary greatly in size, but they all must be able to survive and reproduce to be successful organisms. The cell membrane that envelops its contents must be able to control the movement of nutrients into the cell and of ions, molecules, and proteins out of the cell. Energy is converted from food and/or light and is used to synthesize internal components. The information for reproducing cell structures is encoded within its genetic makeup, thus providing the cell with its own self‐sufficient capability to reproduce life‐supporting needs and to repair genetic damage as needed. When functioning properly, the cell contains all the necessary tools to survive.

1.1.1 Cell membrane

Composition

The cell generally consists of cytoplasm, bounded by a cell membrane, and a nucleus, also enclosed in a membrane. There are exceptions to this model, such as red blood cells that have lost their nuclei during differentiation. The plasma membrane, or cell membrane, defines the boundary of the cell (Figure 1.1) and consists primarily of phospholipids and proteins. The phospholipids form a bimolecular layer, with their hydrophilic ends at the outer surfaces of the membrane and their hydrophobic chains extending into the middle of the membrane. The protein components of the membrane are globular particles distributed through the lipid bilayer; their polar amino acids may be exposed on an outer surface, but nonpolar portions remain in the interior.

Figure 1.1 An electron micrograph showing the various components of a eukaryotic (human) cell.

Physical barrier

The cell membrane serves as a physical barrier for the cell contents, but it is rather fragile. If one were to tear a hole in this membrane by micromanipulation, the contents would spill out into the surrounding medium. An intact cell can rapidly repair minor membrane damage, but more extensive damage leads to cell death.

Regulatory barrier

The membrane also acts as a regulatory barrier for the entry and exit of molecules and particles. This ability to regulate the passing of substances is called selective permeability. Substances can cross the cell membrane by three mechanisms: by free diffusion along a gradient, meaning that substances travel from regions of high concentration to regions of lower concentration; by active transport, which requires energy and moves substances against a concentration gradient; and by enclosure in vesicles that move substances into the cell (endocytosis) or out of the cell (exocytosis). Water can move freely across cell membranes in both directions; it is this property that allows hypotonic solutions (those less concentrated than the inside of the cell) to swell the mitotic cell, thus facilitating chromosome spreading for cytogenetic study.

Glycoprotein functionality

Molecules of glycoprotein (proteins with sugar molecules attached at points along the amino acid chain) exist on the surface of the protein–lipid membrane and sometimes project through it, into the cell. These glycoproteins function in cell adhesion, both to other cells and to culture flask surfaces. Trypsin, a protease (an enzyme that digests proteins), removes these molecules, thereby freeing cells for subculture or harvest. Glycoproteins can be antigenic (e.g., in red cells they determine blood type), and can serve as receptors for viruses, plant agglutinins (e.g., phytohemagglutinin), and hormones. They are further implicated in contact inhibition, a process in which normal cells stop dividing as cultures become confluent. Tumor cells often lose this property and tend to keep growing unchecked in a disorganized fashion when the growth surface is limited. Glycoproteins on the cell surface are also important in cell–cell recognition. If lymphocytes are stripped of their glycoproteins, they no longer accumulate in the lymph nodes.

1.1.2 Cytoplasm

Cytoplasm is the part of the cell within the cell membrane, excluding the nucleus, that consists of water, inorganic ions or molecules, and a variety of organic compounds. In many ways it resembles a colloid, with particles suspended in a continuous gel‐like substance called the cytosol. The cytosol, in turn, contains a cytoskeleton of tubules and filaments, dissolved molecules, and water. Among the inorganic molecules are potassium, sodium, magnesium, and calcium. Trace amounts of many heavy metals are also present, as are bicarbonate and phosphate. Tiny granules can also be seen with a light microscope. These granules have been shown to be a series of vacuolar structures, bound by lipoprotein membranes similar to the cell membrane, with some even further differentiated into a complex system of internal membranes.

The large organic molecules (called macromolecules), which give the cytoplasm its colloidal properties, can be grouped into three main classes: proteins, nucleic acids, and polysaccharides. Each class is a polymer built from different subunits (monomers): proteins are made up of amino acid subunits; nucleic acids are polymers of nucleotides; and polysaccharides are built from sugar monomers. Together, the organelles described below and the cytosol make up the cytoplasm.

Proteins

Proteins carry out several important functions within the cell, including structural support, catalysis of metabolic reactions, and regulation of complex cellular processes. Examples of structural proteins are actin and myosin in muscle, and keratin in hair, nails, and hooves. Regulatory proteins include hormones, growth factors, and receptors.

Polysaccharides

Polysaccharides function as food storage molecules and as structural molecules. The two most important polysaccharide food reserves in higher organisms are starch and glycogen, both of which are polymers of glucose sugar. Structural polysaccharides include cellulose and chitin: cellulose is the major constituent of cell walls in plants, and chitin is found in the exoskeletons of insects and crustaceans.

Lipids

Another important organic molecule, although it is not classified as a macromolecule, is the lipid. Lipids encompass a diverse group of compounds that are all soluble in nonpolar, organic solvents. Included in this class are fats, which are used primarily for energy storage; phospholipids, which are found in cell membranes; sphingolipids, which are especially prominent in the cell membranes of brain and nervous tissue; glycolipids, which are important in the myelin sheath of nervous tissue; steroids, which include male and female sex hormones, bile acids, adrenocortical hormones, and cholesterol; and fatty acids, which are components of energy storage molecules.

Endoplasmic reticulum

Endoplasmic reticulum (ER) is contiguous with the outer membrane of the nucleus. It is the site for folding proteins and assembling large molecules in...