Introduction

to
CYTOGENETICS

Prepared by:

JOHANNA M. PAGAYAO, RMT

 INTRODUCTION

GREGOR JOHANN MENDEL

Mendel was a biologist, meteorologist, mathematician, Augustinian friar and abbot of St. Thomas'
Abbey in Brünn. Mendel was born in a German-speaking family in the Silesian part of the Austrian
Empire and gained posthumous recognition as the founder of the modern science of genetics.
Mendel's pea plant experiments conducted between 1856 and 1863 established many of the rules of
heredity, now referred to as the laws of Mendelian inheritance. he was known as “Father of Modern
Genetics.”

Cytogenetic- involves testing samples of tissue, blood, or bone marrow in a laboratory to look for changes in
chromosomes, including broken, missing, rearranged, or extra chromosomes. Changes in certain chromosomes may
be a sign of a genetic disease or condition or some types of cancer.

The word genetics, coined by William Bateson (1906), is derived from the Latin word genesis. Genetics is the study
of heredity and variation. Heredity refers to the transmission of genetic information, more precisely genes, from
parents to offspring. Variation refers to the difference among the individuals of a species for a character. It may be
due to heredity or environment.

BRANCHES OF GENETICS

Modern genetics is traditionally classified into three branches as transmission genetics, molecular genetics and
population genetics.

(1) Transmission genetics

Transmission genetics deals with the study of passage of characters from one generation to the next. It is studied
mainly through breeding experiments in individual organism. It also encompasses the basic principles of genetics
such as the relationship between chromosomes and heredity, the arrangement of genes on chromosomes, and gene
mapping. It has important practical application in the development of new varieties in plants and animals. As
Mendel pioneered this approach to genetics it is frequently called as Mendelian genetics or classical genetics.

(2) Molecular genetics

Molecular genetics involves the study of the chemical nature of the gene, its structure, organization, and function at
molecular level. It includes the cellular processes of replication, transcription, and translation and also gene
regulation which is the processes of control of expression of the genetic information. The developments in
molecular genetics have lead to breakthroughs in cloning and genetic engineering.

(3) Population genetics

Population genetics is the study of the variation of genes between and within populations of a species and its
changes over time and space. It is important in evolutionary studies, as evolution is just the change in the genetic
makeup of population over time. However, these fields may overlap each other and each organism for example
Drosophila or maize may be studied at the level of transmission, molecular and population genetics.

IMPORTANCE AND APPLICATIONS OF GENETICS

Genetics plays a vital role in the field of medicine as many diseases and disorders have a hereditary component.
Diagnosis of several genetic disorders which may be expressed in later stages of growth can now be studied in new
born babies.

Examples:

- Genetic disorders
o sickle-cell anemia
o Huntington disease
- Common diseases
o Asthma
o Diabetes
o Hypertension

EARLIER CONCEPTS OF HEREDITY

 - The general aspects of heredity were known to Assyrians and Babylonians centuries before the Christian
era. The nature of heredity of traits has been understood and the genetic principles have been applied in the
domestication of crops such as wheat, peas, lentil, barley and animals like dogs, goats and sheep. This had
lead to the development of agriculture and fixed human settlements. As early as 700 B.C, Babylonians and
Assyrians developed date-palm varieties that differed in fruit color, size and ripening time. They noted that
female palm trees produced fruits only when their flowers were dusted with pollen and that the pollen
parent had a marked effect on the quality of fruits produced by female plants. This effect is known as
metaxenia. Around 4000 BC, the Chinese attempted to improve rice varieties.

- Majority of biologists at the time of Mendel believed, in the theory of spontaneous generation, that primitive
organisms originated from non-living matter e.g., decaying organic matter which was disproved by the
experiments of Redi (1621 – 1697) and Spallanzani (1729 –1799)

1. Pre-formation theory of Swammerdam

- According to pre-formation theory, the egg or sperm contains the miniature copy of adult called
homunculus, which later enlarges during development. It implied that all traits would be inherited from only one
parent. Although many observations suggested that offspring possess a mixture of traits from both parents, pre-
formationism remained a popular concept throughout 17th and 18th centuries.

2. Epigenesis concept of Wolff

Wolff (1738 –1794) proposed that during early stages of development the embryonic tissues are composed of cells
similar in structure and function which later differentiate into adult tissues and organs. This concept is known as
Epigenesis.

3. Inheritance of Acquired characters or Lamarckism

Lamarck (1744 –1829) proposed that characters acquired by individuals of one generation are transmitted to those
in the next generation. According to this theory, if a person develop strong muscles by physical exercise, his children
will inherit this character. The early idea of the inheritance of acquired characters was initially suggested by
Hypocrates (400 B.C.).

4. Pangenesis theory of Charles Darwin

Charles Darwin proposed the concept of pangenesis according to which the particles called gemmules or pangenes
carry information from various parts of the body to the reproductive organs which are transmitted through the
gametes to its progeny and will produce a similar modification in the corresponding organ of the progeny. This
concept though incorrect, persisted from the time of ancient Greeks till 18th century. He believes that small particles
(pangenes) derived from parent were incorporated in tissue of offspring.
5. The Germplasm Theory or Weismannism

August Weismann proposed the germplasm theory and explained that the cells in the reproductive organs carry the complete set
of genetic information that is passed to the gametes and hence the changes taking place in the germplasm are transmitted to the
next generation. The germplasm theory believes that only gonads contribute to offspring.

- Castle and Philip transplanted the ovary of the black guinea pig into the albino guinea pig. They mated the ovary
transplanted albino pig with albino pig, as male. All the progeneies had black fur. If the theories of inheritance of acquired
character and pangenes were correct, all the progenesis of the above mating must be albino, but all were black colored.

6. Blending Inheritance

Biologists before Mendel believed that the medium for inheritance is liquid, possibly blood and that the offspring are a blend of
both the parents. This suggests that the genetic material itself blends and once blended the genetic differences could not be
separated out in the future generation. The fact that the offspring possessed the characters of both the parents strengthened the
blending type of inheritance. Later this theory was disproved by Mendel.

7. Hybridization in plants

Grew (1641-1712) reported that plants reproduce sexually by using pollen grains which induced many botanists to effect crossing
in plants and create hybrids. Kolreuter (1733-1806), foremost among these early plant breeders, conducted extensive study on
hybridization in tobacco. He noted uniformity and heterosis in F1 generation and appearance of variation in F2 generation. As he
crossed plants that differed in many traits, Kolreuter was unable to discern any general pattern of inheritance.

o Thomas Fairchild - produced the first artificial hybrid in plants called ‘Fairchild’smule’ by crossing Carnation and Sweet
William in the year 1717.
o John Knight (1759-1835) - developed commercial varieties of apple, pear, apricot, and grapes through hybridization.
o Amici (1830) - entry of pollen tubes into the ovary.
o Strausberger (1884) - fusion of sperm and egg nuclei i.e., fertilization had clarified the role of male and female gametes in
sexual reproduction.

Developments in cytology in the 1800s had a strong influence on genetics.

o Robert Brown - described the nucleus in 1833.

o Matthias Jakob Schleiden and Theodor Schwann - proposed the concept of the cell theory in 1839, according to which, all
life is composed of cells, cells arise only from preexisting cells, and the cell is the fundamental unit of structure and
function in living organisms.
o Charles Darwin - recognized that heredity was fundamental to evolution and he conducted extensive genetic crosses with
pigeons and other organisms. In 1856, he put forth the theory of evolution through natural selection and published his
ideas in “On the Origin of Species”. However, his lack of understanding on the nature of inheritance became a major lapse
in his theory of evolution.
o 1882 - Walter and Flemming observed the division of chromosomes and coined the word ‘mitosis’.
o 1885 - Waldeyer coined the term ‘chromosomes’ and also it was generally recognized that the nucleus contained the
hereditary information.

8. Particulate inheritance of Mendel

Gregor Johann Mendel began his experiments on garden peas in 1857 presuming that the hybrids will have blending of the parental
traits. Instead, for all the seven traits studied, he observed that the hybrids showed only one of the two parental traits. Mendel’s
results clearly disproved the blending mechanism of inheritance and suggested a particulate theory of inheritance.

The science of genetics founded by Mendel reached the present status by the contribution of many scientists which are
tabulated as follows.
 CYTOLOGY

The cell is the fundamental, structural and functional unit of living organisms. In 1665, Robert Hook observed the presence of cells
in cork tissue. The scientific study of cell is called as cytology whereas; cytogenetic refers to the branch of biology which deals with
the study of chromosomes in relation to genetics.

Shape, size and number of cells

✔ Generally the shape of the cell correlates with its function. For example epithelial cells are flat while muscle cells are
elongated. The cells may be spherical, cylindrical, tubular, hexagonal or irregular in shape. Mostly the cells are cylinders
of 15-30 μ in diameter. The size of the cells ranges from 0.2-0.5 μ in bacteria to 75mm in case of Ostrich eggs. The number
of cells in an organism varies from one (bacteria, protozoans) to trillions (human beings).
CELL THEORY

The German microscopists, Matthias Schleiden and Theodor Schwann proposed the Cell theory in 1839, which states that all
plants and animals are constructed from small fundamental units called cells, and that all cells arise from preexisting cells.
Table -3.1. Differences between prokaryotes and eukaryotes

CHROMOSOME MORPHOLOGY

Strausberger in 1875, first observed thread-like structures during cell division. Waldeyer first used the term chromosome in 1888.
The study of structure and function of chromosomes is termed as Cytogenetic. A chromosome is a rod shaped, self replicating, and
organized mass of DNA present in the nucleus of the eukaryotic cell, visible only during metaphase of cell division. Prokaryotes
have naked circular DNA in their nucleus.

CHROMOSOME NUMBER

All organisms have a characteristic number of chromosomes in their body cells referred to as somatic chromosome number or
diploid (or 2n) number. The somatic cells normally will have two copies of the chromosome which are identical in gene content,
arrangement and morphology and hence are called as homologous chromosomes. One member of each pair is acquired from the
gamete of one of the two parents.

The gametes pollen grains, sperms, egg cells, have only half of the somatic chromosome number termed as gametic chromosome
number or haploid (n). The term ‘ploidy’ refers to the sets of chromosome in the cells. Somatic cells have two sets of chromosomes
and are called diploid (2n). Gametes or sex cells contain only half the somatic chromosome number and are called as haploid (n).
Fusion of the male and female gametes gives rise to diploid zygote and the offspring will get half the chromosome from the mother
and half from the father.

A genome refers to a set of chromosomes corresponding to the haploid set of a species and is represented by X. The term
“haplodiploid” refers to the presence of both haploid and diploid chromosome number in an organism.

CHROMOSOME SIZE

Chromosomes are generally measured during mitotic metaphase as they are very thick and spread well in the cell. It may be as
short as 0.25 µm in fungi and birds, or as long as 30 µm in Trillium. The giant chromosomes in diptera are 300 μ in length and 10
μ in diameter and are visible even in interphase nucleus. In general, plants have longer chromosomes than animals. The organisms
with less number of chromosomes contain comparatively large-sized chromosomes than the chromosomes of the organisms
having many chromosomes. Further, the chromosomes in a cell are never alike in size, some may be exceptionally large and others
may be too small. Stretched end-to-end, the DNA in a single human diploid cell would extend over 2 meters.

CHROMOSOME MORPHOLOGY

A mitotic metaphase chromosome have the following components namely, chromatids,

centromere, telomere, secondary constriction with satellite and chromomere. The short arm is
called as ‘p’ arm while the long arm is ‘q’ arm. The regions and bands are numbered from the
centromere out. to identify a band in the chromosome, a sequence of four items are used
namely, chromosome number, arm, region and band number. For example, 9q34 refers to
chromosome 9, the long arm, region 4 and band 4.

(1) Chromatid: The longitudinal sections of a metaphase chromosome are called as

chromatid. The chromosome at interphase and telophase is represented by a single
chromatid where each chromatid is composed of a single DNA double helix. A
metaphase ,chromosome has two chromatids held together at centromere (Fig. 4.1).

(2) Centromere: A centromere is the constricted region of chromosome which separates it into a short arm and a long arm
(q). The short arm of the chromosome is denoted by p while the long arm is denoted by q (Fig. 4.1). After DNA replication, the
chromosome consists of two identical structures called sister chromatids which are held together by centromere. The
centromere, known as primary constriction, is the point of attachment for spindle microtubules. Before cell division, a protein
complex called kinetochore assembles over centromere on each side of the chromosome to which the spindle microtubules later
attach. As a result centromeres are the first part of chromosome to be pulled towards the poles during
anaphase. Hence, chromosomes without centromere cannot be pulled into newly formed nuclei and are
lost causing catastrophic damage to the cell.

Depending on the position of the centromeres, chromosomes can be grouped as: 1. Metacentric:
Centromere is located at the centre of the chromosome. Here, both arms are equal in size and appear as
‘V’ shape during anaphase. 2. Submetacentric: The centromere is located on one side of the centre
point such that one arm is longer than the other. These chromosomes appear as ‘j’ or ‘V’ shaped during
anaphase. 3. Acrocentric: Centromere is located close to one end of the chromosome forming a very
short arm and a very long arm. They appear as ‘j’ or rod shaped during anaphase and 4. Telocentric:
Centromere is located at one end of the chromosome so that the chromosome has only one arm. These
chromosomes always appear ‘I’ shaped or rod shaped during anaphase. Centromeres are the last segment to replicate during S
phase. As the position of centromere remains fixed in a chromosome, except for structural chromosomal aberrations, they are
used for identification of different chromosomes of a species.

(3) Telomere: The two ends of linear chromosomes are known as telomeres. They help in stabilizing the ends of chromosomes.
If a chromosome breaks, producing new ends, these ends have a tendency to fuse together and the chromosome id degraded at
the newly broken ends. Telomeres provide stability to chromosomes.

(4) Secondary constriction: The constricted or narrow region other than the centromere is called as secondary constriction.
Chromosome may have secondary constriction in one or both arms. Chromosomal end distal to the secondary constriction is
known as satellite or sat-chromosome. The production of nucleolus is associated with secondary constriction. Therefore, it is
also called Nucleolus Organizer Region (NOR) and the satellite chromosomes are often referred to as Nucleolus Organizer
Chromosomes (NOC).
(5) Chromomere: Chromosomes in pachytene stage of meiosis show small bead like structures called chromomeres. They are
regions of tightly folded DNA which are invisible during mitotic metaphase. The distribution of chromomeres in chromosomes is
highly characteristic and constant whereas, the pattern of distribution differs for different chromosomes. They are clearly visible
as dark staining bands in the giant salivary gland chromosomes. Chromomeres may differ in size in a single chromosome of maize
or they appear uniform in size as in rye.

HETEROCHROMATIN AND EUCHROMATIN

The chromosomes are composed of thread like structures called chromatin. Based on its affinity to basic dyes (acetocarmine or
feulgen) during prophase the chromatin was classified into two groups namely heterochromatin, and euchromatin.

In general heterochromatin is found in centromeric and telomeric regions which generally replicate later than the euchromatic
regions of chromosomes. Heterochromatin is further classified into two groups:

a) Constitutive heterochromatin: They are genetically inactive present near centromere and telomere. They do not revert to
euchromatic state and is present at identical positions on both homologous chromosomes of a pair.

b) Facultative heterochromatin: They are genetically active region present in the middle of the chromosome between
centromere and telomere. They play a active role in transcription. A well-known example of facultative heterochromatin is the
Barr body, an inactivated X chromosome in somatic cells of mammalian female (XX).

KARYOTYPE AND IDEOGRAM

The complete set of chromosomes, in the cells of an organism, arranged and numbered by
size, from largest to smallest. its karyotype. It is most often studied when the cell is at
metaphase of mitosis and all the chromosomes are present as dyads.

The general morphology, including size of chromosome, position of centromere, presence

of secondary constriction and size of satellite bodies, of somatic chromosomal
complement of an individual constitutes its karyotype.

The karyotype of the human female contains 23 pairs of homologous chromosomes (22 pairs of autosomes and a pair of X
chromosomes). While, the karyotype of the human male contains the same 22 pairs of autosomes, one X chromosome and one Y
chromosome

Karyotypes are used identify chromosomal alterations that may result in a genetic
disorder. It is also useful to confirm the presence of specific species and to study genetic
diversity in species with a wide range.

The dia-grammatic representation of the karyotype of species showing all the

morphological features of chromosomes is known as ideogram or ideotype.

MOLECULAR STRUCTURE OF CHROMOSOME

Prokaryotic chromosomes consist of a single DNA molecule which is usually circular, with only small amount of associated
proteins. Each prokaryotic chromosome has a single origin of DNA replication. Eukaryotes have several linear chromosomes and
the DNA is tightly associated with large amount of proteins. Each eukaryotic chromosome has multiple origins of DNA
replication.
FOLDED FIBER MODEL (Du Praw, 1965)

Du Praw proposed that the chromosomes are made up of chromatin fibers of about 230
– 300 Ǻ diameter and that each chromatin fiber consists of a DNA double helix in coiled
state. Thus the 300 Ǻ chromatin fiber is produced by coiling of a single DNA double helix
which replicates during interphase, producing two sister chromatin fibers (Fig. 5.1).

As the 0.3 % of DNA remains unreplicated during S phase, the two sister fibers remain
joined in the centromeric region until its replication in zygotene phase.

The folding of the two sister chromatin fibers, reduces their length and increases their thickness and stainability, and gives rise
to two sister chromatids. DuPraw’s model of DNA- histone association is now considered wrong by the discovery that DNA itself
is looped around histone beads to form nucleosomes.

NUCLEOSOME-SOLENOID MODEL (Kornberg and Thomas, 1974)

Chromosomes are composed of chromatin fibers. The term chromatin refers to the mixture of DNA and proteins that composes
the chromosomes. The proteins are of two types : histone proteins and non-histone proteins.

Histones are proteins with a high proportion of positively charged amino acids (lysine and arginine) which enable them
to bind firmly to the negatively charged DNA double helix. The quantity of histones in chromatin is equivalent to the quantity of
DNA in the chromatin, in dry weight basis. All the species and tissues have five types of histone proteins namely, H1, H2a, H2b, H3
and H5. The amino acid sequence of the histones is highly conserved throughout evolution suggesting the importance of their role
in the survival of eukaryotes. DNA coil around the histones to form nucleosome.

Nucleosome is a beadlike subunit of chromatin made up of histone octamer (two

molecules each of H2a, H2b, H3 and H4). This bead like histone octamer is wound twice by
146 bp of DNA. A molecule of H1 histone seals the DNA with the octamer. The nucleosomes
now resemble beads on a string. The section of DNA between the two nucleosomes is called
as linker DNA. The length of the linker DNA varies with species and in humans, it is about
60bp long. These forms the first level of coiling of DNA.

The string of nucleosomes coils further to form 30 nm thick helical structure called
solenoid, which forms the second level of coiling. Solenoid fiber can be seen in electron micrographs of metaphase chromosome.
The solenoid structure further supercoils to form 300 nm loop like structure. This is followed by formation of 700nm rosette
structure (consisting of six connected loops), which then coil finally to form the metaphase chromosome with two chromatids.
DNA packed into solenoids, unlike DNA in nucleosome form, is not transcriptionally active.

The organization of these structures involves the binding of chromatin fibers to chromosomal scaffolding by the non-
histone protein, topoisomerase II. The non-histone proteins are also involved in regulation of gene expression.

Each eukaryotic chromosome is composed of a DNA double helix. The various structures
and their packing ratio are given in table.

SPECIAL TYPES OF CHROMOSOMES

1. GIANT CHROMOSOMES OR POLYTENE CHROMOSOMES

To increase cell volume, some specialized cells undergo repeated rounds of DNA
replication without cell division (endoreduplication). As a result each chromosome
becomes a bundle of numerous chromatids all aligned lengthwise. Being thick and 200 times longer than somatic chromosomes
they are called as giant chromosomes or polytene chromosomes and the condition is known as
polyteny.

Polytene chromosomes were originally observed in the larval salivary glands of Chironomus
midges by Balbiani in 1881. They are known to occur in secretory tissues of other dipteran
insects such as the Malpighian tubules of Sciara and also in protists, insects, plants and
mammals, and hence are called as salivary gland chromosomes. The diffused uncoiled regions
of the polytene chromosome, called as chromosome puffs, are sites of RNA transcription. A
large chromosome puff is called as a Balbiani ring.
Uses

Polytene chromosomes have characteristic light and dark banding patterns. Dark banding frequently corresponds to inactive
chromatin, whereas light banding is usually found at areas with higher transcriptional activity. Any change in chromosome
structure is reflected on the banding pattern. Hence, genetic research involving deletion, inversion, duplication or translocation
can be identified by observing changes in the banding pattern.

2. LAMP BRUSH CHROMOSOMES

They are a special form of giant chromosomes found in the growing oocytes of most animals, except
mammals, visible even in light microscope. They are first seen by Flemming in 1882. Chromosomes
transform into the lampbrush form during the diplotene stage of meiotic prophase I due to an active
transcription of many genes. They are highly extended meiotic half-bivalents, each consisting of 2 sister
chromatids which are organized into a series of chromomeres. From each chromosome, pair of loops
emerges in the opposite directions, vertical to the main chromosomal axis. These lateral loops give these
chromosomes the appearance of a lampbrush, and hence are called as Lampbrush chromosomes.

Amphibian and avian lampbrush chromosomes can be microsurgically isolated from oocyte nucleus
(germinal vesicle) with either forceps or needles. A given loop always contains the same DNA sequence, and it remains extended
in the same manner as the oocytes grows. These chromosomes produce large amounts of RNA for the oocyte, and most of the
genes present in the DNA loops are being actively expressed.

Uses

Giant chromosomes in the lampbrush form are useful model for studying chromosome organization, genome function and gene
expression during meiotic prophase, since they allow the individual transcription units to be visualized. Moreover, lampbrush
chromosomes are widely used for high-resolution mapping of DNA sequences and construction of detail cytological maps of
individualchromosomes.

3. ACCESSORY/SUPERNUMERARY CHROMOSOMES

In many species, some extra chromosomes are found in addition to normal somatic chromosomes. These extra chromosomes are
called accessory chromosomes. They have some peculiar functional aspects. In some animal species, they may arise due to
fragmentation of heterochromatic Y chromosome. These chromosomes are generally smaller in size than the normal somatic
complement. They are believed to be generally inactive genetically. In many species they tend to be eliminated from somatic
tissues due to lagging and non-disjunction and they frequently change their morphology through fragmentation.