Chromosome Techniques

Archarna Sharma

• Publisher: CRC Press
• Year: 2020
• Print ISBN: 9783718655137
• eBook ISBN: 9781000159479
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

This laboratory manual covers the study of chromosomes in plants, animal and human systems, dealing with the protocols and principles involved. It caters to the requirements of scientists working laboratories, presenting details of the operational mechanism for use at the chromosome level.

CHAPTER 1

PRETREATMENT, HYPOTONIC TREATMENT, FIXATION AND STAINING

PRE-TREATMENT AND HYPOTONIC TREATMENT

Pre-treatment for the study of chromosomes is generally performed for several special reasons. It may be carried out for: (a) clearing the cytoplasm, (b) separation of the middle lamella causing softening of the tissue, or (c) bringing about scattering of chromosomes with clarification of constriction regions. Pre-treatment may also be needed to achieve rapid penetration of the fixative by removing undesirable deposits on the tissue as well as for the study of the spiral structure of chromosomes. The first two applications involve removal of extranuclear contaminants, whereas the third and most important one exerts a direct effect on the chromosomes.

Pre-Treatment for Clearing the Cytoplasm and Softening the Tissue

In order to clear the cytoplasm from its heavy contents, acid treatment has often been found to be very effective. Short treatment in normal hydrochloric acid or other acids brings about transparency of the cytoplasmic background. Such treatments require thorough washing for the removal of excess acid and acid soluble materials. However since acid treatment affects the basophilia of the chromosomes mordanting may become necessary after such treatment. It hampers banding patterns as well.

In addition to acids, several enzymes have been applied for clearing the cytoplasm and cell separation through digestion. Most common ones used are pectinase, cytase from snail stomach and also ribonuclease. A complex enzyme preparation ‘Clarase’ gave brilliant preparations following staining. In the authors’ laboratory, treatment with cellulase has given very satisfactory preparations in difficult dicotyledonous materials.

The use of enzymes for cell separation has been dealt with in the chapters on tissue culture and mammalian chromosomes.

Alkali solutions as pre-treatment agents are effectively employed for materials with heavy oil content in the cytoplasm since alkalis remove the oil by saponification. Such solutions commonly employed are sodium hydroxide or sodium carbonate. As in acid treatment, thorough washing in water is necessary after the tissue has been kept in alkali solution.

In certain cases, pre-treatment may be necessary to remove deposits of secretory and excretory substances from the surface of the tissue which may hinder the access of the fixing fluid. The best example is the application of hydrofluoric acid to remove siliceous deposits prior to fixation in bamboos; or a very short treatment in Carnoy’s fluid, containing chloroform, to remove oily or other secretory deposits on the cell walls before fixation, in a number of plant materials.

Separation of Chromosome and Clarification of Constrictions

The underlying principle is the viscosity change in the cytoplasm. As spindle formation is dependent on the viscosity balance between cytoplasmic and spindle constituents, a change in cytoplasmic viscosity brings about a destruction of the spindle mechanism with the chromosomes remaining free or, more precisely, not attached to any binding force within the cell. Pressure applied during squash or smear scatters chromosomes throughout the cell surface. Changes in cytoplasmic viscosity simultaneously affect the chromosome too, which undergoes differential hydration in its segments, and due to this differential effect, constriction regions in chromosomes appear well clarified. A number of pre-treatment chemicals are available, colchicine being the most important one.

Pre-treatment also aids in securing a high frequency of metaphase stages through spindle inhibition. Colchicine is the most active substance known, and also compounds having similar properties, such as chloral hydrate, gammexane, acenaphthene, vinblastine sulphate (Velban) and vincaleucoblastine, amongst others (see table 1 for list).

The concentration used and the period of treatment have to be strictly controlled, as also the temperature in most cases. Prolonged treatment leads to narcotic effects, including chromosome breaks. For further details, see Sharma and Sharma (1980).

Colchicine

(C₂₂ H₂₅ O₆ N) was first isolated from the roots of Colchicum autumnale by Zeisel in 1883. The general method of extraction is through the use of ethanol and subsequent dilution with water, finally the aqueous solution is extracted with chloroform and crystals of colchicine are obtained along with the solvent. The chemical structure of colchicine shows that it has a three-ringed configuration.

Colchicine is the methyl ether of an enolone containing three additional methoxy groups and acetylated primary amino group and three non-benzenoid double bonds. The threshold regions of colchicine-mitotic activity are identical for both crystalline and amorphous forms. Colchicine is soluble in water (500.00 in 10^-6 mol/1).

Though highly water-soluble, it is very active at an extremely low concentration. It falls under Ferguson’s second category of compounds and the reaction is chemical. It brings about a change in the colloidal state of the cytoplasm, causing spindle disturbance.

With regard to the exact reactive groups in the colchine molecule (a) at least one methoxy group in ring A is necessary for its action (b) ring C must be 7-membered and the hydroxyl group should preferably be replaced by an amino group; (c) esterification of amino group in ring Β increases the activity; and (d) isocolchicine and its derivatives are less active. A proper distance should be maintained between esterified side chains of rings Β and C. A number of sulphydryl poisons, such as iodoacetamide, dimercaptopropanol (BAL), mercaptoethanol, sodium diethyldithiocarbamate act in the same way as colchicine.

In the study of chromosomes, without inducing polyploidy, colchicine has to be applied in a low concentration, such as 0.5 per cent for 1 h, thereby straightening the chromosome arms to allow a thorough study of the constriction regions. It is especially effective for long chromosomes.

Colchicine has the added advantage of being active within a very wide range of temperature. The period necessary for the manifestation of effect varies in different plant and animal groups. The range of concentrations is also wide, between 0.001 and 1%. In animal cells the method of application is preferably by injection or by addition to culture medium 2 h prior to harvesting, whereas in plant tissue it is applied through soaking, plugging and injection. It can also be used in lanolin paste and in agar. In artificial culture colchicine is added to the medium. To avoid toxicity affecting other metabolic processes, it is generally applied in a low concentration to animal cells. In general, the drug is exceptionally suitable for the study of chromosome structure and metaphase arrest, provided that strict control is maintained over the concentration and period of treatment.

Colcemid (Ciba) is de-acetyl methyl colchicine and is used more extensively in studies of mammalian chromosomes since it does not show certain toxic effects attributed to colchicine.

Acenaphthene, a naphthalene derivative has the same property as colchicine of arresting metaphase though its use is limited. As its structure is quite different from that of colchicine, several other aromatic compounds were tried and various derivatives of benzene and naphthalene were found to be effective.

Chloral hydrate

[C Cl₃ C Η (OH)₂] has been used as a ...

Table of contents

• Cover
• Halftitle Page
• Title Page
• Copyright Page
• Table of Contents
• PREFACE
• INTRODUCTION
• CHAPTER 1 PRE-TREATMENT, HYPOTONIC TREATMENT, FIXATION AND STAINING
• CHAPTER 2 MICROSCOPY, MICROSPECTROPHOTOMETRY, FLOW CYTOMETRY, IMAGE ANALYSIS, CONFOCAL MICROSCOPY
• CHAPTER 3 STUDY OF BANDING PATTERNS OF CHROMOSOMES
• CHAPTER 4 AUTORADIOGRAPHY—LIGHT, FIBRE AND HIGH RESOLUTION
• CHAPTER 5 ELECTRON MICROSCOPY
• CHAPTER 6 STUDY OF CHROMOSOMES FROM TISSUE AND PROTOPLAST CULTURE, CELL FUSION AND GENE TRANSFER IN PLANTS
• CHAPTER 7 CHROMOSOME ANALYSIS FOLLOWING SHORT- AND LONG-TERM CULTURES IN MAMMALIAN AND HUMAN SYSTEMS, FROM NORMAL AND MALIGNANT TISSUES
• CHAPTER 8 SOMATIC CELL FUSION IN ANIMALS
• CHAPTER 9 EFFECTS OF EXTERNAL AGENTS AND MONITORING FOR ENVIRONMENTAL TOXICANTS
• CHAPTER 10 ISOLATION AND EXTRACTION OF NUCLEI, CHROMOSOMES AND COMPONENTS
• CHAPTER 11 MICRURGY
• CHAPTER 12 IDENTIFICATION OF CHROMOSOME SEGMENTS AND DNA SEQUENCES BY IN SITU MOLECULAR HYBRIDIZATION
• CHAPTER 13 SPECIAL MOLECULAR TECHNIQUES NECESSARY FOR CHROMOSOME ANALYSIS
• CHAPTER 14 REPRESENTATIVE SCHEDULES FOR DIRECT OBSERVATION OF CHROMOSOMES FROM PLANTS AND ANIMALS IN VIVO AND FROM SPECIAL MATERIALS
• REFERENCES
• INDEX