Karyotype analysis, Banding patterns, & Karyotype evolution,

KARYOTYPE ANALYSIS AND BANDING PATTERNS

Karyotype Analysis

A group of plants or animals comprising a species is characterized by a set of chromosomes, have certain constant features. These features include chromosome number, size and shape of individual chromosomes and other attributes listed above. The term karyotype is given to the group of characteristics that identifies a particular chromosome set and is usually represented by a diagram called ideogram where chromosomes of haploid set of an organism are ordered in a series of decreasing size. The karyotypes of different groups are sometimes compared and similarities in karyotype are presumed to represent evolutionary relationships.

Karyotype also suggest primitive or advanced feature of an organism.

A karyotype showing large differences between smallest and largest chromosome of the set and having fewer metacentric chromosomes, is called asymmetric karyotype, which is considered to be a relatively advanced feature when compared with symmetric karyotype. A symmetric and an asymmetric karyotype are shown in figure.

In 1931 G. A. Levitzky, a Russian scientist suggested that in flowering plants there is a predominant trend towards karyotype asymmetry. This trend has been carefully studied in the genus Crepis of the family compositae. In several cases it was shown that increased karyotype asymmetry was associated with specialized zygomorphic flowers.

The karyotype of the human female contains 23 pairs of homologous chromosomes:

• 22 pairs of autosomes
• 1 pair of X chromosomes

The karyotype of the human male contains:

• the same 22 pairs of autosomes
• one X chromosome
• one Y chromosome

A karyotype is a technique that allows geneticists to visualize chromosomes under a microscope. The chromosomes can be seen using proper extraction and staining techniques when the chromosomes are in the metaphase portion of the cell cycle. Detecting abnormalities is important for prenatal diagnosis, detection of carrier status for certain genetic diseases or traits, and for general diagnostic purposes.

1.  Sterilization-It is an important step.All instruments and equipments should be properly sterilized.

2. Sampling and Culture- Karyotype analysis can be performed on virtually any population of rapidly dividing cells either grown in tissue culture or extracted from tumors. Chromosomes derived from peripheral blood lymphocytes are ideal because they can be analyzed three days after they are cultured. Lymphocytes can be induced to proliferate using a mitogen (a drug that induces mitosis) like phytohemagglutinin. Skin fibroblasts, bone marrow cells, chorionic villus cells,tumorcells, or amniocytes also can be used but require up to two weeks to obtain a sufficient amount of cells for analysis.

1.      Cell Synchronization- The cultured cells are treated with colcemid, a drug that disrupts the mitotic spindle apparatus to prevent the completion of mitosis and arrests the cells in metaphase.

2.      Harvesting and Slide preparation- The harvested cells are treated briefly with a hypotonic solution. This causes the nuclei to swell making it easier for technicians to identify each chromosome. The cells are fixed, dropped on a microscope slide, dried, and stained.

3.      Observation in microscope - The most common stain used is the Giemsa stain. Other dyes, such as fluorescent dyes, can also be used to produce banding patterns.

4.      Photography- Chromosome spreads can be photographed.

5.      Enlargement of photo and rearrangement to form Karyotype- The photographs are enlarged, cut out, and assigned into the appropriate chromosome number or they can be digitally imaged using a computer. In case of Human karyotype, there are seven groups (A-G) that autosomal chromosomes are divided into based on size and position of the centromere. The standard nomenclature for describing a karyotype is based on the International System.

Karyotype Evolution

Two aspects of the process of speciation are of interest in the context of cytogenetics. The first of these is changes in ploidy i.e. changes in the number of the chromosomes, which themselves remain unaltered. Changes in ploidy can have both genetic and phenotypic effects, such as fertility changes, and can be used to great effecting plant breeding to produce new cultivars. Additional sets of chromosomes can be from the individual (autopolyploid) or from an organism of genetically distinct origin (allopolyploids).In the case of humans, changes in ploidy can have very severe consequences, as can the second process of interest: changes in karyotype.

Karyotype changes can be thought of as being due to changes in either DNA content or chromosome structure as well as changes in chromosome numbers. In this context it is possible to see speciation and karyotype changes that are linked, as in the marsupials, or not linked, as in the hominids.

Evolution and speciation are closely related to observable changes in an organism’s chromosomes. It should, however, be clearly borne in mind that karyotype changes are rarely enough for speciation to occur on their own. It is, after all, the phenotype expression of the genome which determines the position of the fine line, sometimes indefinable, between variation and speciation.

Banding Patterns

Introduction

 Karyotype analysis is a technique where chromosomes are visualized under a microscope. Cells are collected from an individual, induced to divide, and then arrested at metaphase (a stage of cell division when the chromosome are condensed and therefore visible). The chromsomes are stained with certain dyes that show a pattern of light and dark bands, which is called as the banding pattern. These bands reflect regional differences in the amounts of A and T versus G and C. The banding pattern for each chromosome is specific and consistent allowing identification of each of the chromosomes.

Preparation of chromosomes for karyotype analysis can be performed in a number of ways and each will yield differing pieces of information. The chromosomes may be stained with aceto- orcein, feulgen or a basophilic dye such as toluidine blue or methylene blue if only the general morphology is desired.
If more detail is desired, the chromosomes can be treated with various enzymes in combination with stains to yield banding patterns on each chromosome. These techniques have become common place and will yield far more diagnostic information than giemsa stain alone (the most commonly used process). A band is an area of a chromosome which is clearly distinct from its neighboring area, but may be lighter or darker than its neighboring region. The standard methods of banding are the Q, G, R, and C banding techniques. These are defined as follows:

1. G-Banding  

This is so called because of the use of Giemsa stain. The bands stained with Giemsa were designated G bands. This is a simple two step process which involves a pretreatment followed by staining with the stain which may be either Giemsa, leishman’s stain or Wright stain. Pretreatment involves any treatments which remove or damage the protein content but not the DNA of the chromosome. This may be either acid hydrolysis with methanol-acetic acid or enzymatic treatment with trypsin or pronase enzymes. Enzymatic treatment is preferred.

Giemsa is a complex stain based on methylene blue and eosin, it is a reaction that stains the chromosome.

G bands are richer in A&T about 3.2%. During cell cycle, light bands in interbands replicate earlier, followed by the dark G bands.

2. Q-Banding

 Q Banding utilizes quinacrine dihydrochloride stain and so named. This intercolates between the DNA and fluoresces when UV light falls on it.

 Identification of individual chromosomes can be made 1^st time by the use of fluorescent Q-banding which produces similar banding pattern that of G-bands except it use fluorescent dyes, like proflavine l quinacrine and especially an alkylating derivative, quinacrine mustard. This banding led to the discovery of a sub-structure along the length of the chromosomes.

Some of the alternating stains are acridine orange, EtBr, propidium iodide and Hoechst 33258 and chemically most of these are based on structure of multiple benezene rings. Multiple fluorochromes can be used simultaneously to increase the apparent contract which in turn enhance the banding pattern and so the result.

The major advantage of using fluorochromes for staining is that the bonding between dye and DNA is electrostatic and therefore is easily reversible. So the DNA sample can be restained for more accurate results. Other advantages are that these stains are relatively less toxic so can be used in cell culture. They are more sensitive so can detect DNA even if concentration may be very low.

Fluorochrome banding has some drawbacks –

1.      High resolution Q-Banding is not practicable because sharp image cannot be produced.

2.      High cost of equipment required for fluorescent microscopy, as UV light bleached the fluorochromes.

3.      Toxic nature of fluorochromes, so handling required great care.

Both Q and G bands correlate with the centromeres observed in leptotene and pachytene chromosomes during meiosis. Q and G banding pattern are generally similar and correspond to the intercalary heterochromatin.

3. C-Banding

This banding is used to identify the regions of constitutive heterochromatin which is genetically inert highly repetitious DNA sequences like satellite DNA. C-Banding is species as well as individual specific.

It is also a two step process i.e. pretreatment followed by staining. Pretreatment involves treatment with hydrochloric acid followed by either NaOH or Barium hydroxide treatment, and then incubation in a warm salt wash, and then finally Giemsa staining. In this, HCl depurinates the DNA without breaking the sugar –PO₄²-bonds. This is stopped before the degradation of the entire DNA. Then by denaturation, the alkali treatment helps in solubilization of DNA which is further aided by warm salt wash treatment which breaks the sugar-phosphate backbone.

4. R-Banding  Also known as Reverse Banding as the banding pattern obtained is the reverse of that found with the Q and G banding. R-bands contain all of the housekeeping genes of the cell and about half of the tissue specific genes which are rich in GC and have CpG islands while dark G bands are rich in AT (Reverse).

R-bands can be produced either by incubating chromosomes in a hot saline buffer, followed by staining in Giemsa, or by staining heat treated chromosomes directly with acridine orange.

This technique is particularly useful in diagnostic cytogenetics when examining telomeric deletions which, with G-banding are normally light staining and difficult to quatify.

The nature of the production of R-bands is thought to be related to the base content of DNA.G-bands are AT rich,and AT rich DNA denatures at lower temperature than GC-rich R-bands.After the heat treatment the G-bands would be denatured,but the R-bands are still double stranded,and the nature of the staining reflects this situation.

5. Brd U Banding

 In this method, Brd U, an analogue of thymidine is applied which get incorporated into chromosome during s-phase of cell cycle. This incorporate of Brd U changes the staining properties to UV and washes in hot buffered saline, as Brd U inhibit Giemsa staining so the areas of chromosome having Brd U is less stained as compared to other parts.

Brd U Antibodies or other similar analogoues like bromodeoxycytidine can also be used instead of Giemsa for detection.

It has two methods –

i) B-pulse,                                   

ii) T-pulse,

6. RE Banding

As the name suggest, it involves use of restriction endonucleases for banding.

7. D-Banding

Produced due to differential sensitivity of chromatin to enzyme DNase I therefore also referred as DNase I sensitivity banding.

8. NOR Banding

This method was successfully used in the Cytogenetics Laboratory of the Laboratory of Pathology in Seattle, Washington, USA.

Chromosomes are treated with silver nitrate solution which binds to the Nucleolar Organizing Regions (NOR), i.e., the secondary constrictions (stalks) of acrocentric chromosomes.

9. T-Banding

T-banding is used to stain the telomeric regions of chromosomes for cytogenetic analysis. Telomeric (or terminal) banding was first reported by Dutrillaux, who used two types of controlled thermal denaturation followed by staining with either Giemsa or acridine orange. The T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric. (Gustashaw, 1991).

10. DAPI/Distamycin A Staining

The DAPI/Distamycin ,a staining technique is useful in identifying pericentromeric breakpoints in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques. Also, DAPI/DA is the method of choice for Yqh chromsome material in suspected Y autosome translocations.

The DAPI/distamycin,a fluorescent staining technique was first described by Schweizer, Ambros, and Andrle as a method for labeling a specific subset of C bands. (Gustashaw, 1991 ).

History of Chromosome Banding Techniques
Stain or Banding Technique	Investigator	Year
Q-banding	Caspersson, Zech, Johansson	1970
G-banding (by trypsin)	Seabright	1971
G-banding (by acetic-saline)	Sumner, Evans, Buckland	1971
C-banding	Arrighi, Hsu	1971
R-banding (by heat and Giemsa)	Dutrillaux, Lejeune	1971
G-11 stain	Bobrow, Madan, Pearson	1972
Antibody bands	Dev, et al	1972
R-banding (by fluorescence)	Bobrow, Madan	1973
In vitro bands (by actinomycin D)	Shafer	1973
T-banding	Dutrillaux	1973
Replication banding	Latt	1973
Silver (NOR) stain	Howell, Denton, Diamond	1973
High resolution banding	Yunis	1975
DAPI/distamycin A stain	Schweizer, Ambros, Andrle	1978
Restriction endonuclease banding	Sahasrabuddhe, Pathak, Hsu	1978