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1.

CELL STRUCTURE
All organisms are made of cells. They are the building blocks.
Definition of a Cell
A cell is the fundamental and simplest integrated and continuously changing unit of the
structure and function of an organism delimited by a semi permeable plasma membrane
and capable of self – reproduction.
Types of Cells
Based on this definition, there are two types – (1) the prokaryotic cells and (2) the
eukaryotic cells.
Viruses
Viruses are exceptions. They are primitive. A virus is neither a cell nor an organism. They
are not complete cells.
COMPARISON OF PROKARYOTIC AND EUKARYOTIC CELLS
SNo Characters Prokaryotic Cell Eukaryotic Cell
1 Size Small Large
1 - 10µm 10 - 100µm
2 Plasma membrane Present Present
3 Mesosome Present Absent
4 Cell wall Present – made of Present in plant cells – made
amino sugars & of cellulose
Muramic acid
5 Cytoplasm Streaming movement Streaming movement
absent present
6 Mitochondria Absent Present
7 Golgi body Absent Present
8 Lysosomes Absent Present
9 Endoplasmic reticulum Absent Present
10 Vacuoles Absent Present in plant cells
11 Ribosomes Small – 70 S Large – 80 S
12 Photosynthetic Chlorophyll-a, etc. Chloroplasts with
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machinery chlorophyll- a & b in plants


13 Nucleus Absent Present
14 Nucleoid Present Absent
15 Nuclear membrane Absent Present
16 Nucleoplasm Absent Present
17 Chromosome 1, circular, More, thread-like, with DNA
with DNA
18 Genetic material DNA DNA
19 DNA Circular Linear
20 Flagella Present – no 9+2 Present – with 9+2 pattern
pattern
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2. BACTERIA
(Prokaryote Cell)
Introduction
Bacteria are prokaryotes. They are small, microscopic, simple, unicellular and most
primitive. From them arose the eukaryotes.
General features of Prokaryotes
A prokaryotic cell is a one-envelope system. It has the central nuclear components. They
may be DNA, RNA and nuclear proteins. The nucleoli are absent. They are not covered
by membrane. They are surrounded by cytoplasm. The cytoplasmic organelles like
endoplasmic reticulum, Golgi complex, mitochondria (the respiratory enzyme system),
Centrioles, basal bodies, cytoskeleton, etc. are not well defined. The nuclear
components and cytoplasm are together covered by a plasma membrane.
Ultra Structure
The structural details of a bacterium can be seen only under the electron microscope.

I. Outer Covering
Bacteria have an outer covering of three layers. They are,
a. Plasma Membrane
The protoplast of bacteria is covered by the plasma membrane. It is living and dynamic.
It is ultra thin (6 to 8 nm).
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Chemical Structure
Plasma membrane is made of lipid and protein molecules. These macromolecules are
arranged in a particular pattern. It is called the fluid mosaic pattern.
Fluid Mosaic Pattern
Lipids are united with phosphates. They are called phospholipids. They are present in
two layers. They are polar. They have a head-end and a tail end. The tails are fatty-acyl
chains. The polar heads are on the surface. The tails face the interior.
The protein molecules are embedded within this bi-layer. They may be on the outer,
middle or inner sides.
Functions of Plasma Membrane
The membrane proteins act as carriers or permeases. They selectively transport
substances in and out. Some proteins are involved in oxidative metabolism. They act as
enzymes and carriers for electron flow. It provides attachment for the circular
chromosome (DNA).
Intrusions
There are two main types of intrusions (infoldings) of the plasma membrane.
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1) Mesosome or Chondrioid
This is an inward extension. It is made of convoluted membranes. It forms complex
whorls. It increases the surface area and enzymatic contents. Mesosomes are present
in chemoautotrophic and photosynthetic bacteria.
2) Chromatophores
They are seen in photosynthetic bacteria. They are membranous. They bear
photosynthetic pigments. They have different forms – vesicles, tubes, bundled tubes,
stacks or thylakoids.
B. Cell Wall
The cell wall is outer to plasma membrane. It is strong and rigid. It is chemically
different from the cell wall of plants. It has proteins, lipids, polysaccharides and chitin,
and not cellulose.
Ultra structure of the Cell Wall of Gram-negative Bacteria
It is thicker, amorphous, homogeneous and single-layered. It contains the following
chemicals: peptidoglycons, proteins, neutral polysaccharides and polyphosphate
polymers. It is made of two layers. They are,
a) Gel, Proteoglycon or Peptidoglycon
This is situated around the plasma membrane. It contains periplasmatic space.
b) Outer Membrane
It consists of a lipid bilayer.The lipids are phospholipids and lipopolysaccharides. In it are
many channels of porin polypeptide. Solutes diffuse through these channels.
C. Capsule
This is the outermost covering. It is a layer of slime or gel. It is thick, gum-like and
mucilaginous. Plasma membrane secretes it. It protects and regulates water and ion
concentration.
2. Cytoplasm
The cytoplasm is covered by the plasma membrane. It has two parts – 1) hyaloplasm
and 2) matrix or cytosol.
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Cytosol
This is the ground substance. All metabolic activities take place here. It consists of
water, proteins, lipids, carbohydrates, different types of RNAs and many smaller
molecules.
The cytosol is differentiated into two regions. They are 1) the nuclear area and
cytoplasmic area. The nuclear area is less electron dense (light). The cytoplasmic area is
electron thick (dark). The second area has two types of inclusions. They are Ribosomes
and Reserve materials.
Ribosomes
There are 1000s of ribosomes. They are 25 nm in diameter. They are made of RNA and
proteins. The type of these ribosomes is 7oS. It is made of two subunits. One is larger.
It is the 50S subunit. The other is smaller. It is the 30S subunit. Some ribosomes are
non-functional. They are separated as subunits. Protein synthesis takes place in them.
During protein synthesis, many ribosomes unite to form polyribosomes.
Reserve Materials
They are stored in the cytoplasm. They are finely distributed or distinct granules. The
second is also called inclusion bodies or storage granules. There are three types of
reserve materials.
i) Organic Polymers
They serve as carbon reserves and energy reserves.
ii) Volutin
They are more in quantity. They are the reserves of inorganic phosphate. They are
highly refractive.
iii) Sulpher
It is formed from the oxidation of hydrogen sulphide. They are spherical droplets. They
are energy reserves. They are seen in some sulpher bacteria.
3. Nucleoids
The Nucleoid is a specific and clear region of the cytoplasm. The nuclear material is
present here. It is not separated from the cytosol by membrane. It includes the nuclear
material. It is the single, circular and double-stranded DNA. This is called the bacterial
chromosome. It is permanently attached to the plasma membrane at one point.
Histone proteins are absent from them.
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There is only one species of RNA polymerase. It forms all the three types of RNAs –
mRNA, rRNA and tRNA.
4. Plasmids
They are extra-chromosomal genetic materials. They are small, circular and closed DNA
molecules.
5. Flagella and other structures
Flagella
Many bacteria are mobile. One or more flagella do this function. The flagella are
smaller and simpler than the eukaryotic flagella. They are 15 to 20 nm in diameter.
Their length is upto about 20 µm.
Structure
The flagellum has a helical tube. It has a single type of protein subunit called flagellin.
The tube is attached to a base through a hook. This is short and flexible. It can be
rotated. A ‘motor’ does this.
The flagellum has three parts. They are tube, hook and base.
a. The tube is helical. It is formed of a single type of protein subunit called
flagellin.
b. The hook joins the tube and the base. It has a rotatory motor. It rotates
like the propeller of a ship.
c. The motor is formed of four parts – rotor (M ring), stator, bearing (S
ring) and rod. The rotor is a protein disc. It is embedded in the plasma
membrane. Using energy, it rotates rapidly. The stator forms the base
for rotation. It is also a protein disc. The rotor is attached to the hook
and flagellum by a rod. When the rotor rotates, the flagellum also
rotates. The rod passes through the outer membrane of the cell wall.
At that point, there is a hole. The bearing seals this hole.
The number and arrangement of flagella in bacteria has four types.
Monotrichous - A single flagellum at one end.
Lophotrichous - Many flagella at one pole.
Amphitrichous - At lease one flagellum at each pole.
Peritrichous - Flagella all over the surface.
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Axial Filaments
This is seen in some spirochetes. They move like snakes. They do not project out. They
lie on the cell surface.
Fimbriae or Pili
Some bacteria like the Gram-negative bacteria have this. They are non-flagellar, very
fine appendages. They do not move. They help in attachment. It also helps
conjugation. Bacteriophages attach to the bacteria on the pili. The plasmid genes code
them.
Spinae
They are tubular, pericellular and rigid appendages. They are formed of the protein
spinin. They are seen in some Gram-positive bacteria. They aid in tolerating pH,
temperature salinity, etc.
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3. EUKARYOTIC CELL
Introduction
The eukaryotic cells are two-envelope systems. There are secondary membranes around
the nucleus and other cell organelles. In the cytoplasm, they form the endoplasmic
reticulum.
The eukaryotic cells are true cells. They occur in plants and animals. They are typically
composed of plasma membrane, cytoplasm and its organelles and a true nucleus. The
nucleus is separated from the cytoplasm by a thin, perforated nuclear membrane.
General Structure of a Eukaryotic Cell
Shape
The basic shape is spherical. The function of the cell determines the shape. So, the
shape may be fixed or variable or irregular. Plasma membrane, exoskeleton, surface
tension, viscosity of the protoplasm, cytoskeleton, mechanical action of the nearby cells,
etc. also determine the shape.
The cell shape varies from animal to animal, organ-to-organ and even in the same
organ.
The different shapes are: polyhedral (many-sided), flattened, cuboidal, columnar,
discoidal, spherical, spindle-shaped, elongated, branched, etc.
Size
The eukaryotic cells are typically larger. They are larger than the prokaryotic cells. They
range from 10 to 100 µm. The biggest single cell is that of the unicellular organism,
Amoeba proteus. It is 1000 µm. The single-celled alga, Acetabularia, is unusually long –
10 cm. In multicellular organisms, the ostrich egg is the biggest – 18 cm.
Cell Volume
The cell volume of each cell type is mostly constant. It is not related to the size of the
organism. This is called the law of constant volume.
Ratio of Volume to surface
This should be within a limited range. Large cells have proportionately large volume and
less surface area. Small cells have proportionately less volume and large surface.
Metabolically active cells are smaller in size.
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Cell Number
This varies from organism to organism. Unicellular organisms have one cell. In
multicellular organisms, the number of cells is related to the size of the animal. Large
animals have more cells. Small animals have less number of cells. An 80 kg man may
have about 60 thousand billion cells. Though the number of cells is indefinite, it may be
constant also (ex.: Rotifer). This phenomenon is called eutely.
Ultra Structure of a Typical Animal Cell
The typical parts of an animal cell are
• Plasma membrane,
• Cytoplasm and
• Nucleus.

1. Plasma Membrane or Cell Membrane or Plasmalemma


This forms the cell boundary. It is a living, thin and delicate membrane. It is tri-laminar
or 3-layered. The outer layers are dark. The middle layer is translucent.
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Its molecular structure shows a bimolecular lipid layer. Protein molecules are
embedded or attached to its surfaces. Some carbohydrate molecules may also be
attached to lipids (glycolipids) or proteins (glycoproteins). This is called unit membrane
structure.
Plasma membrane is selectively permeable. It controls and selects the entry or exit of
materials. Due to this, the cell environment is maintained constant (homoeostasis).
This transport of materials may be active (using energy rich ATP) or passive (osmosis,
diffusion, etc). In the first type, the carrier molecules (membrane proteins) play an
important role. They are called transport proteins or pumps.
Bulk entry of large molecules is called endocytosis. Bulk exit of large molecules is called
exocytosis.
The cell organelles like mitochondria, Lysosomes, Golgi bodies, endoplasmic reticulum
are membrane-bound. These membranes are similar to plasma membrane.
2. Cytoplasm
Cytoplasm is inner to the plasma membrane. It is differentiated into the following.
Cytosol or Matrix
It is a colloidal organic fluid. Outside the nucleus, it is called the cytoplasm. It fills all the
spaces of the cell. It forms the internal milieu. It determines many fundamental
properties of cells. Many small molecules for cellular metabolism are dissolved or
suspended in it. It contains the soluble proteins and enzymes. The cytosol is
differentiated into an outer, non-granular, viscous, clear and rigid ectoplasm or cell
cortex and an inner, granular and less viscous endoplasm.
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Cytoskeleton

Inside the cytosol, there are many fibers. They are collectively called the cytoskeleton.
They give shape, mobility and attachment to other cell structures. The fibers are of
three types – microtubules, microfilaments and intermediate filaments.
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Microtrabecular Lattice
This is a three-dimensional network. The cytoskeletal fibers are interlinked. It is flexible
and can change its shape. The cell organelles are attached to it.
Cytoplasmic Structures
Cytoplasm includes two types of structures. One type is living and the second is non-
living. They are suspended in the cytoplasm.
Non-living or Cytoplasmic Inclusions or Paraplasm
They are the stored food and secretory substances. They are in the form of refractile
granules. They include oil drops, triglycerols, yolk granules, secretory granules, glycogen
granules, etc.
Living or Cytoplasmic Organelles
There are many types of organelles. They are membrane-bound. They do specialized
tasks.
Endoplasmic Reticulum
This is a living cytoplasmic organelle. It is in the form of a wide reticulum (network). It
has membrane-bound channels. Some portion of it is attached with the plasma
membrane and the nuclear membrane. At some surface areas, ribosomes are attached.
This gives a rough appearance. So, this is called the Rough Endoplasmic Reticulum
(RER). The other surface areas are called Smooth Endoplasmic Reticulum (SER). The
SER’s functions are lipid metabolism, glycogenolysis and drug detoxification.
The RER participated in photosynthesis. The produced proteins enter into the lumen of
the endoplasmic reticulum. There, they rearrange, mature and become secondary and
tertiary proteins. It also produces cellular membranes.
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Golgi apparatus

This is a living cytoplasmic organelle. It is in the form of a cup. It is membrane-bound.


It is near the nucleus. It is formed of many cisternae. They are smooth. They are
closely packed together in parallel rows. Spherical vesicles surround them. They are
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also membrane-bound. They are of three types. In active cells, they are well formed
and many.
Functions
Golgi apparatus
a. stores secretory materials like enzymes, mucin, melanin pigment, etc.,
b. processes proteins,
c. synthesizes polysaccharides and glycolipids,
d. sorts and sends proteins to the various parts of the cell,
e. adds membranous elements and
f. forms the acrosome of the sperm.
Lysosomes
Lysosomes are living cytoplasmic organelles. They are tiny, spherical (polymorphic) and
membrane-bound. The Golgi apparatus forms them. They contain about 50 hydrolytic
enzymes. These enzymes digest intracellular and extra-cellular materials. They have an
acidic medium (pH 5).

Ribosomes
Ribosomes are living cytoplasmic organelles. They are tiny and spherical. Their
diameter is from 150 to 200 Å. They contain RNA and proteins. They are the ‘work-
benches of protein -synthesis’. They may be free or attached to the endoplasmic
reticulum. Their sedimentation quotient is 80S (40S + 60S).
Mitochondria
Mitochondria are living cytoplasmic organelles. They are ribbon-shaped. Two
membranes surround each mitochondrion. The inner one contains many enzymes,
coenzymes and electron. transport particles. It projects inward as finger-like cristae.
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The mitochondrial matrix is a colloid. It contains enzymes used in Kreb’s citric acid
cycle.
Mitochondria are the seat of oxidative phosphorylation.

This produces energy as ATP molecules. So, mitochondria are called the powerhouses
of the cell. They produce part of their needed proteins. So, they are also called the
semi-autonomous organelles.
Cytoplasmic Vacuoles
They are many, small or large, hollow and liquid-filled. They are formed from the Golgi
apparatus or endoplasmic reticulum. A lipoprotein membrane surrounds them. Their
function is storage, transmission of materials and internal cell pressure maintenance.
Peroxisomes
These are small and surrounded by membrane. Inside are some enzymes. They
participate in detoxification activities, degradation of amino acids and β-oxidation of
fatty acids.
Microtubules and Microtubular Organelles
This is seen in almost all eukaryotic cells. They are fibers 24 nm in diameter. Its wall is
thick. It is made of α-tubulin and β-tubulin protein subunits. The center is hollow. The
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microtubules can be assembled or dissembled by adding or removing protein subunits.


Cilia, flagella, basal bodies and centrioles are formed from the microtubules.
3.Nucleus
The nucleus is in the center of the cell. It is round. It controls all the vital activities of
the cytoplasm. It carries the hereditary material, the DNA.

The nucleus can be differentiated into the following parts.


Nuclear Membrane
There are two nuclear membranes – an inner and an outer. The outer membrane is a
continuation of the endoplasmic reticulum. The nuclear membranes have pores, the
nucleopores.
Nucleoplasm
This fills the nucleus. It is surrounded by the nuclear envelope. It contains materials
needed for DNA replication, transcription, etc.
Nucleolus
The nucleolus is a sub-organelle. It is round and prominent. It stains dark. It has no
limiting membrane. It is formed by the ribosomal DNA (rDNA). This, in turn, is formed
by the nuclear organizer. Nucleolus forms the ribosomes.
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Chromatin
The chromatin carries the genetic or hereditary units, the genes. The chromosomes
form a network, the chromatin network. The chromatin has two parts, the euchromatin
and the heterochromatin. The first is genetically active. The second has no genetic role.
The chromatin contains one DNA molecule.
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4. CELL DIVISION
Introduction
The cells vary widely in structure and function. But they have some important properties
in common. Plant cells differ from animal cells in shape etc. Both cells are essentially
alike in terms of genes, chromosomes and other structures and functions.
Cell division is a process of reproducing itself. The function of the cell division is an
inherent property of the cell to produce identical cells. This leads multicellular
organisms to grow. Cell division is really a process of duplication or multiplication.
W.flemming(1843-1915) accounted the mechanism of cell reproduction. It involved two
inter related process,viz;
a. Mitosis, the nuclear division.
b. Cytokinesis.
MITOSIS
Division of a cell begins after a more or less protracted period known as interkinesis or
interphase . In the interphase nucleus, the chromosomes are not visible but they
appear as long , thin ,intertwined threads. In the intetphase accounts for about 90% of
the entire cell cycle . in the interphase stage , the cell grows and makes copies of the
chromosomes. The interphase has three phases,viz;
1. G - 1 phase: This is the first part of the interphase. At this phase the cell grows.
2. S phase: It is the synthesis part of the interphase. Here the chromosome IS
copied.
3. G - 2 phase: It is the second part of the interphase. The result of the interphase
is the duplication of DNA. At the end of the of the interphase, the chromatin
containing DNA becomes invisible.
[Chromatin which contains all DNA of nucleus in plant or animal cell - that later
condense to form the choromosome]. Nucleolus and centrioles are formed.
The remaining part of the cell cycle, has 4 phases – Prophase, Metaphase, Anaphase
and Telophase.
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PROPHASE
• The nuclear membrane breaks down, and eventually disappears.
• Chromosomes threads (chromatin) begin to coil up. Chromatin is formed into
chromosome. The chromosomes shorten and thicken.
• Centrioles separate and start to move away
• Spindle fibres begin to form

Metaphase:
• It is the longest stage of the mitosis.
• The centrosomes move to the opposite sides of the cell.
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• The mitotic spindle apparatus is formed out of the centrosomes.


• The chromosome are lined up along the equatorial plate of the cell. They are
attached to the mitotic spindle apparatus.
• The chromosomes are composed of two chromatids each.

Anaphase:
• It is the shortest stage of the mitosis.
• The newly formed chromatids are pulled along the spindle fibres towards the
opposite poles from the equator.
• Centrosome splitting marks the beginning differentiation of chromatids into
chromosome.
• Chromosome move apart to the opposite sides of the cell, as the cell grows
• It results in each side of the cell having same number of the chromosomes.
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Telophase:
• Nuclear membrane formed around the chromosomes.
• Chromosomes at the opposite poles begin to uncoil and become less dense.
• The indentical nuclei are fully formed.

Cytokinesis:
Cytoplasm slits in two to form 2 daughter cells. In anaimal cytokinesis , a cleavage
furrow is caused at the surface of the cell membrane . The cleavage furrow is made out
of the actin microfilaments. Their furrow forms into ring. It further deepens and
squeezes the cell into 2 identical cells.
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Mitotic Apparatus
The cytoplasmic organelle, the centrioles forms a part of a large and elaborate structure
called the mitotic apparatus. The centrioles are not observed in-vivo in most cells. It is
more often surrounded by a clear zone – ‘microcentrum or centrosome’ and then by a
denser zone – ‘centrosphere’. From the centrosphere, the asters radiate.
The term mitotic apparatus is applied to the assembly of structures that forms the
achromatic figure in the mitosis. This structure includes the asters [astropheree] which
surround the centrioles and also the mitotic spindle. The aster appears in fixed
preparations as a group of radiating refringent fibrils that converge towards the
microcentrum and continue in the centrosphere. The aster is also evident in-vivo
because of its refringence, but in this case the fibrillar structure of the aster is not seen,
and its make-up seems homogenous.
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The following classes of spindle fibres are observed through the light microscope;
• Chomosomal fibres – joining the poles to the kinetochores of the chromosomes
• Continuous fibres – extending from pole to pole.
• Astral fibres and
• Interzonal fibres.
Role of the mitotic apparatus:
The role of the spindle in the movements of the chromosomes during anaphase is still
amazing. The contraction and shortening of the spindle fibres appear to take place at
the early anaphase. Other evidence accounts that at anaphase, the two sets of
daughter chromosomes are pushed apart by an elongation of the spindle fibres in
between the poles. This 2 types of anaphase movements are concerned with the
chromosomal fibres and the continuous fibres. This two types of movements may occur
in more or less in different proportions according to the cell type.
The equilibrium dynamic model of Inoue explain the process of spindle action.
According to this model, the equilibrium between a large pool of monomers and the
oriented polymers form the spindle fibres or microtubules. Upon polymerization, some
water molecules are believed to dissociate. It aids in contraction and elongation of
fibres. The contraction and elongation of fibres are responsible for the regular
movements of chromosomes during cell division. The contraction and elongation of
fibres is due to the subtraction or addition of monomers from the polar regions. This
leads to consequent reduction of fibre length and pulling of chromosomes towards the
pole / consequent elongation of fibre length and pushing of chromosomes towards the
pole.
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5. Meiosis
Introduction
The reduction in chromosome number from diploid to haploid is done by the specialized
cell divisions of meiosis. Cells prepare for meiosis by replication of their genetic material
as in mitosis. However, instead of a single division as in mitosis, meiosis consists of two
consecutive divisions.
The first meiotic division
In the first meiotic division, the number of cells is doubled but the number of
chromosomes is not. This results in reduction in chromosome number into half per cell.
Prophase I
The prophase I is important stage in meiosis. In this phase the 5 sub stages are seen.
They are Leptotene, Zygotene, Pachytene, Diplotene and Diakinesis. During these stages
the chromosomes behave characteristically.
• The chromosomes first become visible as thin threads within the nucleus at the
stage called leptotene.
• In the next phase, zygotene, the homologous pairs of chromosomes become
closely associated along their lengths. This process is called synapsis [or] joining.
This formed a structure comprised of two chromosomes, a bivalent.
• The bivalents shorten and thicken throughout the next stage pachytene.
Synapsis is complete and the bivalents are held together throughout their
length by a structure known as the synaptenemal complex.
• In diplotene stage crossing over occurs. At this point, portions of one chromatid
of one chromosome may become exchanged with the corresponding region
from a non-sister chromatid of the other chromosome in the pair.
• The chromosomes continue to condense. The synaptonemal complex breaks
down. The homologous chromosomes appear to repel each other. They remain
held together only at chiasmata, the points where crossing over have occured,
and at the centromere. This phase is named diplotene
• There is always at least one chiasma per chromosome arm. Some of the longer
arms will have two or even three. This chiasma HOLDS the chromosomes
together until they separate in anaphase.[ At this point the oocyte enters
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meiotic arrest where it will remain for many years until it is reactivated at
ovulation.]
• The final stage of prophase is diakinesis in which the nuclear membrane breaks
down. The chiasmata slip to the ends of the chromosome arms.
• Centrosomes begin moving to the poles of cell.
Metaphase I
• The bivalents align at the cetre of the cell.
• They are attached to the spindle fibres.
Anaphase I
• The first division begins.
• Each bivalent divides so that one chromosome moves to one pole and the
second chromosome moves to the other. [ Each chromosome is still comprised
of two chromatids.]
• The homologous chromosomes separate

Telophase I
This stage may be absent in some species.
The second meiotic division
• The second meiotic division is simply like mitosis. The number of chromosomes
does not get reduced.
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• Between I and II meiotic divisions, there occurs interkinesis similar to the


interphase of mitosis. It differs in respect to the non-synthesis of DNA.
• In males this follows immediately on the heels of the first division with no
intervening round of DNA synthesis. Prophase II, Metaphase II and Anaphase II
resemble mitosis but with only a haploid chromosome number.
• The second meiotic division in the egg is not completed until fertilization. It is
again very unequal giving the mature egg and a small second polar body
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6. Nucleic Acid – DNA - STRUCTURE


Introduction
James Watson, Francis Crick, Maurice Wilkins and Rosalind Franklin played a role in
discovering the structure of DNA, deoxyribonucleic acid — the molecule that encodes
genes in all living things.

The nucleic acid, DNA, is a double stranded poly – nucleotide structure. The nucleotides
are made of a nitrogenous base, a pentose sugar - a ribose and a phosphate.
Nitrogenous bases
The nitrogenous bases are five types. They are grouped as purines and pyrimidines
based on their structure. The purines are adenine and guanine having two ringed
structures. The pyrimidines are cytosine, thymine and uracil. Thymine is present in
DNA. Thymine is replaced by uracil in RNA.
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Pentose Sugar
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Both DNA and RNA contain pentose sugar. In RNA, the sugar component is ribose, as
indicated by the name "ribonucleic acid". In DNA or deoxyribonucleic acid, the sugar
component is deoxyribose. The prefix “deoxy” means that an oxygen atom is missing
from the second carbon atom of the ribose sugar.
Nucleoside
When a sugar bonds together with a nitrogenous base, it is known as a nucleoside. The
nitrogen base binds with the first carbon atom of the sugar. As such four nucleosides
are known for DNA viz. adenosine, guanosine, cytidine and thymidine. In RNA,
thymidine is replaced by uridine.
Nitrogenous Base + Pentose sugar = Nucleoside

Nucleotides
The binding of phosphate at the fifth carbon of the sugar in the nucleoside forms the
nucleotide. The chemical bond formed between the sugar and phosphate is an ester
bond. The nucleotides are referred as adenylic acid, guanilic acid, cytidic acid and
thymidilic acid. In RNA, thymidilic acid is replaced by the uridilic acid.
Nucleoside + Phosphate = Nucleotide
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Chargaff’s Rule [Chemical equivalence rule]


Erwin Chargaff's work showed certain relationships between the amounts of various
bases. These relationships are called Chargaff's rules. They are
• The amount of adenine equals the amount of thymine.
• The amount of guanine equals the amount of cytidine.
• The amount of adenine plus guanine equals 50% of the total implying that 50%
of the bases in DNA are purines.
• The amount of thymine plus cytosine equals 50% of the total implying that 50%
bases in DNA are pyrimidines.
Chargaff documented that base compositions actually varied among species, that is, in
all species, [A] = [T] and [C] = [G].
The ratio [C+G] / [A+T] are typically less than unity (that is, [C+G] is less abundant).
Maurice Wilkins, and Rosalind Franklin Work
Work on x-ray diffraction patterns by Maurice Wilkins and Rosalind Franklin revealed
that the nucleic acid molecule has a "helical shape" with repeating distances of 0.34 nm,
2nm diameter between the strands and 3.4 nm intervals between the base pairs.
James Watson and Francis Crick model of DNA Double Helix
• Watson and Crick constructed a DNA model based on the findings of Wilkins and
Franklin. In that, the nitrogenous bases are facing the interior of the double
helix. They accounted that adenine can form two hydrogen bonds with
thymine. Cytosine forms three hydrogen bonds with guanine. The Watson &
Crick Model explained the basis of Chargaff’s rules. It is a well-established fact
that where an A exists along a DNA strand, there is a T on the opposite strand
and, where there is a C on one strand, there is a G on the partner strand.
• Watson and Crick then connected base pairs with phosphodiester bonds that
spaced the bases 3.4 Å apart and rotated each subsequent base pair by 36°.
This rotation forms a right-handed double helix with 10 bases per turn and
repeating elements with every 3.4.
• The backbone of each strand consists of alternating phosphates and
deoxyriboses. To be more specific, the phosphate of a nucleotide bonds both to
the 5' carbon of one deoxyribose and the 3' carbon of the next deoxyribose.
33

This is how the nucleotides create the strand. The 5' and 3' refers to the place
of the carbons in the deoxyribose molecule.
• The heterocyclic nitrogenous bases, which are attached to the deoxyriboses,
project in towards the axis of the helix. The two polynucleotide strands wound
around each other. The strands of a DNA double helix are anti-parallel which
means that one chain runs 5'-3' and the other runs 3'- 5'.
• The joining of the two strands is by the hydrogen bonds between the nucleotide
bases of the two strands. The chemical bind occurs between the adenine and
thymine (2 hydrogen bonds) and between cytosine and guanine
(3 hydrogen bonds). The two strands are complementary to each other. This
means that the strand running in the 5' to 3' direction will have base Adenine
that will pair with the base Thymine on the opposite strand running in the 3' - 5'
direction. Thus the strands are complementary and anti-parallel to each of
them.

• The DNA Double Helix makes a complete turn in over 10 nucleotide pairs, so
each turn takes 34.4 Å. About 25 hydrogen bonds are created in this complete
turn. The power of these 25 bonds is equal to 1 covalent bond (bond between
34

carbon and oxygen). The diameter of a DNA Double Helix is 20 Å. The hydrogen
bonds and the bonds between deoxyribose and phosphates are the main (but
not the only) chemical forces that create a DNA double helix.
35
36

7. CANCER
“Growth for the sake of growth is the ideology of cancer cells.”
Introduction
Cell division results in growth and repair. Sometimes, cells of a particular type divide
rapidly, abnormally and uncontrolled at the cost of other cells. This leads to cancer.
These cells are called the neo-plastic cells. The resulting growth is called the neo-plastic
growth or tumour. The tumours may be benign or malignant.
Benign Tumour
When a tumour is formed as a result of abnormal and continuous cell division restricted
to the site of origin, it is termed benign. These tumour cells will be well-differentiated.
Malignant Tumours
The tumour cells may be carried by the blood or lymphatic circulation to other parts.
They may also directly penetrate other parts. There, they may induce secondary or
metastatic tumours. These tumours are termed malignant. They cause death. They
contain undifferentiated cells. Their nuclei and nucleoli are large.
Types of Cancer
Cancer is actually a bundle of diseases. There are many types. They grouped into four –
carcinomas, sarcomas, lymphomas and leukaemia.
Carcinomas
The cells of these tumours are formed by the epithelial cells of the ectoderm or
endoderm. The tumours will be solid.
Examples: Nerve tissues (brain), tissues of body surface (skin), glands of body surface
(breast), and cervical, etc. carcinomas.
Sarcomas
These solid tumours grow on the mesodermal connective, cartilage, bone and muscle
tissues.
Lymphomas
These tumours are caused by the excessive production of lymphocytes by the lymph
nodes and spleen.
Example: Hodgkin’s disease.
37

Leukemias
Leucocytes or WBCs neo-plastically produce these tumours.
Mixed Malignant Tumours
These are tumours formed jointly by both the ectodermal and mesodermal tissues.
Cancer Cells’ Characteristics
All differentiated cells can become neo-plastic or cancerous. The inability to control its
rate of division and becoming a tumour is called transformation. They retain the basic
characteristics of their types. In addition, they have the following special characteristics.
Immortalization
The transformed cells can survive permanently. They are immortal. But, normal cells
cannot.
Loss of Contact Inhibition
The plasma membranes of normal cells may contact in a culture. They stop movement
and growth. This is called contact inhibition. But, the transformed cells do not have this
property. They grow continuously. The membranes are less adhesive. Even on contact,
they dissociate easily. This helps them to penetrate into other organs.
Reduced Attachment of Cells
Normal cells tend to attach with each other easily. There is specificity in this. That is,
kidney cells attach to only other kidney cells. But, the transformed cells do not attach
with each other easily. They attach with other types of cells too. This helps them to
invade other organs.
Ability to Invade
Transformed cells can invade into other tissues. This may be due to changes in plasma
membrane or release of protease enzymes.
Anchorage Independent
For growth, normal cells need attachment to some substance. The transformed cells do
not need them. This character is used to differentiate a normal and a transformed cell.
Serum Requirement Low
Compared to normal cells, the transformed cells can grow even in media containing less
serum. External substances like serum are needed to lower the levels of intracellular
38

cyclic AMP. This, in turn, induces mitosis. As the transformed cells divide abnormally,
they do not need serum.
Lectins Agglutinate Selectively
Lectins are proteins. In some animals, it is present. It binds to branched, surface-
membrane receptors like oligosaccharides. This results in agglutination/clumping of
cells. This process is less in normal cells and more in transformed cells.
Molecular Changes in Cell Membrane Components
Differences between the surface cell membranes of
Normal Cells Transformed Cells
Four types of Phospolipids Four types of Phospolipids
Form lipid bilayer Form lipid bilayer
Glycolipids and glycoproteins inserted Glycolipids and glycoproteins inserted
4 types of gangliosides – GM1a, GM1, Only GM3
GM2 and GM3
Some major surface glycoproteins
disappear
39

Cytoskeleton Dismantling
The microtubules and microfilaments form the cytoskeleton in normal cells. They are
arranged in a regular fashion. This regulates cell movement. In transformed cells, they
are less and thinner. This is due to polymerization. This results in the movement of
membrane proteins. Blebs, micro-villi and ruffles are also formed.
Membrane Surface Negative Charge Increases
Evident from higher anodic mobility.
Increased Sugar Transport
Due to abnormal growth and multiplication, the tumour cells take in more sugar. This is
seen transported across the membrane.
Immunological Surveillance
Some antigens present in the plasma membranes of cancer cells are not seen in normal
cells. Example: EB Nuclear Antigen. They immunize against themselves. This is called
immunological surveillance. Under favourable conditions, this happens. Only when this
fails, tumours are formed.
Loss of Electrical Communication
The electrical connections between normal cells is absent in cancer cells.
Increase in the secretion of Proteolytic Enzymes
All cancer cells secrete large amounts of proteolytic enzymes.
40

Aldolases
There are three forms of aldolases – Isozymes A, B and C – in mammalian tissues. In
cancer cells, B is replaced by A.
Increase in Glycolysis
In cancer cells, lactic acid is produced more. This is due to glycolysis or anaerobic
respiration. Glucose intake increases.
Hypotheses for the occurrence of Cancer
There are three.
1. The Somatic Mutation Hypothesis
“Somatic mutations in normal cells transform them into cancer cells.”
Some genes are inactivated through the production of repressor proteins. Mutations
may block the production of these repressor proteins and activate the genes. The genes
may be directly mutated also. These lead to alteration in the cell’s control mechanism
and unregulated division or cancer. Role of chromosomal changes in malignant
transformation is inconclusive.
41

2. The Viral Genes Hypothesis


There are evidences that viruses cause cancers in animals. The tumour-producing
viruses are called the oncoviruses.
Virus-Cancer Relationship, some difficulties:
• Risk of injecting cancer-causing viruses into man
• Viruses may be non-transmissible
• Viruses may not have a separate identity
• Virus genetic material may be incorporated into human chromosomes
• Confusion by the presence of harmless viruses.
Evidences:
• Through electron microscopy and immunology
• Epstein-Barr (EB) Virus – Burkett’s Lymphoma
• EB Virus – Nasopharyngeal carcinoma
• Herpes simplex Type 2 Virus – Cervical cancer
• Retoviruses
• Virus-like RNA and DNA particles
• The wart virus – only benign growth.
Incorporation of Viral DNA into the Host Genome
All viruses do not normally grow inside all cells. Each has specificity. Such cells are
called permissive cells. Cells in which viruses do not grow are the non-permissive cells.
Behaviour of Virus on entering host cells
A) Lytic Infection or Productive Infection: They multiply inside the host cells. Kill them.
This is called the lytic phase. Example: Adenovirus.
B) Oncogene Formation: The viral DNA gets inserted and integrated into the host DNA.
The virus is now termed a provirus. This causes cancer. The genes of a virus causing
transformation are called Oncogenes.
Carcinogens
Cancer-causing agents are called carcinogens. The carcinogens affect the normal
genetic processes. Due to this, the cell’s control mechanism is altered.
42

For example, the ears of rabbit were painted with coal tar. It produced carcinomas in
the rabbit. The 3, 4 – benzapyrene, present in the coal tar, was found to be responsible
for this. This shows that the carcinogen induced the carcinoma. Even when the tar was
removed, the carcinomas grew. Its continued presence is not necessary for the growth
of abnormality.
There are many types of carcinogens like Chemicals and Radiations.
Chemical Carcinogens
Mode of Action
Many chemical carcinogens chemically bind with DNA, RNA and proteins and induce
cancer. Mostly, they are chemically transformed into derivatives. These are more
carcinogenic. Example: Nitrates are not mutagenic. But, nitrosamines are powerful
mutagens and carcinogens.
• Chemical carcinogens directly transform cells to malignancy.
• Carcinogens are mutagenic. The DNA sequence is altered.
• Carcinogens can alter gene expression also. This will lead to abnormal
differentiation. Thus, gene expression is altered.
Carcinogenic Induction of Cancer
• Many mutational defects combine to cause cancer.
• Carcinogens or their derivatives bind with DNA. Mutation takes place. Activities
of the genes are affected. Cancer results.
• In some genes, some parts of a gene may code for a harmful effect. They might
be suppressed. The carcinogens remove the suppression. The DNA parts
actively code and produce cancer.
• Oncogene Theory: All cells carry information for malignancy and cancer-causing
viruses (the oncogenic viruses). Carcinogens induce these viruses. They cause
cancer directly or indirectly.
3. The Defective Immunity Hypothesis
Spontaneous mutations occur more. Cancers occur less. This indicates that 1) cancer-
producing mutations are suppressed or 2) the newly produced cancer cells are
destroyed somehow. The second is more possible. This is called immunological
surveillance. This is a case of defective immunity.
43

4) Other Theories
Holley, 1969 – The surface membrane of cells is modified during early transformation.
Due to this, growth and division regulating nutrients (de-oxyglucose) increase inside the
cell.
Comings, 1973 – Cells have structural genes. They code for transforming factors.
Regulator genes suppress them. On removal of suppression, cancer occurs.
Oncogenic Viruses
Viruses causing tumours are called oncoviruses. The following viruses cause it.
1. Papovaviruses
2. Parvoviruses
3. Adenoviruses
4. Herpes viruses
5. Poxviruses
All viruses do not normally grow inside all cells. Each has specificity. Such cells are
called permissive cells. Cells in which viruses do not grow are the non-permissive cells.
Behaviour of Virus on entering host cells
A) Lytic Infection or Productive Infection: They multiply inside the host cells. Kill them.
This is called the lytic phase. Example: Adenovirus.
B) Oncogene Formation: The viral DNA gets inserted and integrated into the host DNA.
The virus is now termed a provirus. This causes cancer. The genes of a virus causing
transformation are called Oncogenes.
Proteins
Early Proteins
Soon after infection, T antigen or tumour-antigen is detected in the cell nucleus.
Interferon inhibits the synthesis of T antigen. Interferon specifically inhibits viral protein
synthesis and not cell protein synthesis. This shows that T antigen is coded by the viral
DNA. Only after this, DNA replicates.
Late Proteins
The capsid proteins are the late proteins. Two of them are identified.
44

Retrovirus
The RNA Tumour Viruses or Oncornaviruses contain RNA and cause cancer. They are
classified as retroviruses. Their RNA forms a DNA copy. This is a reverse method of
forming RNA from DNA. So, they have properties of both RNA and DNA viruses. The
DNA genes are combined with the host’s chromosomal DNA. The virus particles contain
the RNA genes (infectious).
Many retroviruses are found to cause different types of cancers in fish, reptiles, birds
and mammals. Infection in humans is not clear.
The infection of retroviruses is classified as productive and non-productive. In the
former, progenies are produced. In the latter, the progenies are not produced.
In some viruses, a part of the genome may be missing. They are the defective viruses. A
cell may contain one virus. It may not affect the cell. Another virus may also infect it.
This is called super-infection. Due to this, the first virus causes cancer. This second virus
is called the helper virus.
Transmission of Oncornaviruses
Horizontal Vertical
One host to another Parent to offspring.
through contagion. Congenital Genetic
From mother to offspring Through father
through or mother.
ovum/placenta/milk.
45
46

8. MENDELIAN TRAITS IN MAN


The most striking attribute of a living cell is its ability to transmit hereditary properties
from one cell generation to another. Hereditary transmission through the union of
sperm and egg was understood by 1860, and the chromosomes present in the nucleus
were considered as the active factors. The establishment of haploid (n) number of
chromosomes in sperm and egg, during meiosis is significant. This is necessary for
keeping the chromosome number constant as diploid (2n) in the daughter generations.
These merely suggest that chromosomes carry the hereditary information.
GREGOR MENDEL (1822 – l884)

J. Gregor Mendel did his experiments during 1856 -


1864 in garden peas, Pisum sativum. Mendel’s
father had a great love for plants and hence
influenced his son, as they worked together in
garden and orchard. Later Mendel was intensely
interested, in plant hybridization. Mendel’s
experiments, with pea plants were elegant. His
inferences constituted the foundation for the
modern science of genetics. Mendel conducted his
experiments systematically and drew the
conclusions for the data collected. Mendel
presented them in a paper entitled ‘Experiments in
Plant hybridization’ and read before the Brunn
Natural History Society in 1865, and published in the
proceedings of BNH Society. His paper was considered as classic in biology for its
elegance and simplicity.
MENDEL’S CHOICE OF MATERIAL
Mendel was successful because he
• used peas, Pisum sativum, an annual plant which were easily grown and
produced successive generations rapidly.
• selected easily observable 7 characters.
47

• strictly controlled the fertilization process. They have perfect flowers possessing
both male and female parts performing self-pollination. So, cross-pollination
does not occur ordinarily without the investigator’s intervention.
• used mathematics rigorously to analyze his results and to derive good judgment.
• used large numbers of plants
• studied traits that had two easily identified factors.
Mendel examined the following seven characters found in peas:
• Flower colour, purple or white
• Flower position, axial or terminal
• Seed colour, yellow or green
• Seed shape, round/spherical or wrinkled
• Pod shape, inflated or constricted
• Pod colour, green or yellow
• Stem height, tall or short

MENDEL’S METHOD OF CROSSING


Mendel needed to control fertilization. Self-fertilization was ensured by placing a bag
over the flowers to make sure pollen from the stamens lands on the carpel of the same
flower. Cutting off stamens from a flower before pollen was produced, then dusting the
carpel of the flower with pollen from another plant ensured cross-fertilization. To
ensure reliability, Mendel used thousands of plants in each experiment. Mendel worked
48

with true-breeding plants: self-fertilized plants, which produced all offspring identical to
the parents.
Mendel first cross-fertilized two true-breeding plants for one characteristic. For example
tall plants were crossed with short plants (Mendel called these plants a P1 parent
generation). The offspring produced are called F1 (1st filial) generation. The F1
generation were then self-fertilized or cross-fertilized to produce a second generation,
F2
TERMINOLOGIES
Alleles:
The paired factors, held for a single character, are termed as alleles or allelomorphs.
Allele is an alternative form of a gene. Usually there are two alleles for every gene.
Sometimes as many as 3 or 4 [Multiple alleles for a single character].
Example: For the character height in pea plant, the tall plants may have either TT or Tt.
Homozygous:
When the alleles for a particular type of character are same (or) alike, it is termed as
homozygous for that character.
Example: Tall - (TT) and dwarf - (tt) types for character height in pea plant.
Heterozygous:
Here the alleles for a particular type of character are not same or alike. In such cases the
dominant allele is expressed.
Example: Tall-(Tt)
Homologous:
Chromosomes with same genetic loci (or) having similar gene sequences are called
homologous.
Hybrid:
An individual / offspring resulting from a cross between two different contrasting
genomes is termed as hybrid. The heterozygous character is also said to be hybrid.
Phenotypes:
Any measurable (or) visible character of an individual are termed as phenotype. This is
always expressed in words.
Example: Height- Tall/Dwarf & Cotyledon colour - Yellow/ Green
49

Genotypes:
The allelic composition of an individual to distinguish from the physical appearance is
termed as genotype. It is always expressed in alphabets / letters.
Example: Tall - TT/Tt, Yellow - YY/Yy, Dwarf – tt, Green – yy
Normally, the alphabet / letter used will be the first letter of the dominant type of the
character. The capital alphabet / letter for the dominant type and the corresponding
small letter for the recessive type should be used.
Example: Tall (T), Dwarf’(t).
Dominant
A term applied to the trait (allele) that is expressed regardless of the second allele.
Recessive
A term applied to a trait that is only expressed when the second allele is the same (e.g.
short plants are homozygous for the recessive allele).
Pureline:
An individual homozygous for a particular character is termed as pure line or true
breeding variety.
CONCEPTS OF MENDEL
The interpretation of Mendel‘s experiments gave the concept of segregation and
independent assortment of alleles. By segregation, the separation of alleles resulting in
pure gametes is described. The independent assortment explains the independent
combinations of different pairs of alleles during gamete formation. These concepts are
the basic foundation of Mendelian heredity.
PRINCIPLE OF SEGREGATION & MONOHYBRID CROSS
Mendel did his experiments by planting two varieties of Peas, which have two definite
alternate / contrasting traits for a character. He considered the character, height with
one tall type /trait and other with dwarf (short) type. When they were selfed to several
generations, tall never turned into short and short never turned into tall traits. Thus the
consistency of the traits was studied. Mendel called these two traits as true or pure line.
Mendel crossed the true tall and dwarf varieties of garden peas. In the first (F1)
generation, [F Symbolized filial from the Latin, meaning progeny] all plants were tall.
When these F1 tall plants were self pollinated, the missing trait, dwarf re-appeared in
the F2 generation. In this experiment, among the total of 1064 F2 plants, 787 were tall
50

and 277 were dwarf varieties, giving the ratio as 3:1. This type of cross between parents
differing in only one trait or in which only one trait is considered is termed as Mono -
hybrid cross.
Mendel called the trait appeared in the Fl as dominant and its alternative, which was
masked, as recessive.
LAW OF DOMINANCE
In a cross between organisms, which are pure for contrasting traits of a particular
character, only one of the traits appears in the Fl generation. The other trait is masked
or disappeared. The appeared trait is said to be dominant, over the other trait. The
disappeared trait is known as recessive.
The monohybrid cross shall be symbolized as above to explain the segregation of alleles.
Alleles control the expression of traits of a character. Mendel assumed that every
character is determined by some ‘factor’. It is transmitted from parents to the offspring
through the gametes. Mendel’s results of monohybrid crosses accounted the separation
of pairs of alleles resulting in the ‘purity of gametes’. Thus the concept of segregation is
presented as Mendel’s first principle.
LAW OF SEGREGATION
Mendel’s first law or law of segregation, states that the longitudinal separation of the
members of the pair of homologous chromosomes leads to the complete separation (or)
segregation of their allelic genes.
Since every individual is the result of the union of two (opposite) gametes, each
character must necessarily be represented by two factors. In modern days, the factors
are called as genes. Thus, the pure line tall plant and dwarf plants must have two genes
for tallness (TT), and dwarf ness (tt) respectively. Segregation, the separation of the
pairs of genes, occurs in the formation of gametes. Each gamete produced by tall plant
carries only one T gene, likewise t gene in the gamete of dwarf plant. The union of these
gametes resulted in Tt. Because of the presence of T gene in F1, all plants were tall.
While the F1 plants were producing the gametes, half of the gametes carried T gene and
the other half the t gene. The result of selfing the F1 indicated to Mendel that the genes
were entirely separated from each other during gamete formation.
The principle of segregation was further demonstrated, when the F1 plants were
crossed back with the recessive trait. The results showed the separation of paired genes,
by expressing both tall and dwarf in equal proportions among progeny.
51

PRINCIPLES OF INDEPENDENT ASSORTMENT AND DIHYBRID CROSS


Mendel crossed a variety of pea plants having round seeds and yellow cotyledons with
wrinkled seeds and green cotyledons plants. From the previous monogenic studies,
genes for both round and yellow were known to be dominant. Genes for wrinkled and
green seeds were found to be recessive. Hence, as expected, in the cross between pure
line round yellow with wrinkled green, all the Fl progenies were round yellow. The self-
52

pollination of F1 seeds produced 4 phenotypes in a definite ratio. From a total of 556 F2


seeds, 3l5 round yellow, 108 round green, 10l.wrinkled yellow and 32 wrinkled green
were observed. These were found to fit very nearly a ratio of 9:3:3:1.
From the observed results, Mendel inferred the members of pairs of alleles segregate,
and also one pair behaved independently with respect to another pair. Thus the concept
of independent assortment is explained as Mendel’s second principle.
LAW OF INDEPENDENT ASSORTMENT
Mendel’s second law or law of independent assortment states that when a dihybrid
parent produces reproductive cells, the two pairs of alleles (genes) controlling two
different characters must have separated or assorted independently of each other.
Mendel’s dihybrids cross between plants with round - yellow seeds and those with
wrinkled- green seeds shall be represented diagrammatically using the generalized
Mendelian pattern. The pure line Round Yellow parent shall produce only one type of
gamete (RY), carrying one member from each pair of alleles. Similarly, the wrinkled
green parent shall produce one type of gamete (ry), having one member from each pair
of alleles. On fusion of these gametes, the Fl plant will have, RrYy, exhibiting
heterozygosity for both pairs of alleles. The self-pollination of the Fl plant produces four
types of gametes, following the independent assortment of pairs of alleles controlling
two different characters. The random fusion of gametes leads to 16 progenies having 4
phenotypes and 9 genotypes, which was shown in the Punnet checkerboard.
53
54

By using forked line method also, the F2 ratio can be drawn;

BACK CROSS AND TEST CROSS


A cross between F1 organism with either of its parents is termed as back cross. It is
represented as follows;
F1 X Homozygous dominant parent
[OR]
F1 X Homozygous recessive parent
55

But a cross between FI hybrids (or) an organism of unknown genotype with the
recessive organism is termed as test cross. The tests cross helps to determine the
genotype of the unknown organism. If the F1 organism (or) unknown genotype is
crossed with the recessive parent, at least one recessive trait is produced in a series of
offsprings. But in the cross between unknown genotype and the homozygous dominant
trait, it cannot be possible to know the genotype of the organism in question. In
testcrosses, the phenotypic & genotypic ratio will always be equal i.e., 1:1.
Example:

Diagram Showing Test Crosses Involving Monohybrid And Dihybrid Parents


The principle of segregation is also demonstrated well in testcrosses. The result shows
the separation of paired alleles, by expressing all the parental traits in equal proportions
among the progeny. Test cross, is also one of the types of backcrosses, and hence all
testcrosses are backcrosses, but all backcrosses are not testcrosses.
MENDELIAN TRAITS IN MAN
S.No. CHARACTER DOMINANT TRAIT RECESSIVE TRAIT
1. Hair colour Dark Blended / Light
2. Hair type Curly Straight
3. Body hair Intensive Sparse
4. Skin colour Normal / Colour Albinism / White
56

5. Eye colour Dark Light / Blue


6. Eye lashes Long Short
7. Vision Near sighted Normal
8. Vision Long sighted Normal
9. Vision Night blindness Normal
10. Ear lobes Free Attached
11. Hearing & speech Normal Deaf - Mutism
12. Lips Thick Thin
13. Nostrils Wide Narrow
14. Stature Short Tall
15. Fingers and toes Supernumerary Normal
16. Blood groups A,B,AB O
17. Rh factor Positive Negative
18. Blood clotting Normal Abnormal
19. Handedness Right Left
20. Clasping of hands Right over left Left over right
21. Teeth enamel Normal Brown
22. Tongue rolling Able Unable
23. Tongue folding Able Unable
24. Chin Normal Receding
25. Dimple Present Absent
26. Blood clotting [Sex linked] Normal Hemophilic
27. Vision [Sex linked] Normal Colour blindness
28. Hair [Sex linked] Normal Baldness

Pedigree Analysis
A pedigree is a diagram of family relationships that uses symbols to represent people
and lines to represent genetic relationships. These diagrams make it easier to visualize
relationships within families, particularly large extended families. Pedigrees are often
57

used to determine the mode of inheritance (dominant, recessive, etc.) of genetic


diseases. A sample pedigree is below.

In a pedigree, squares represent males and circles represent females. Horizontal lines
connecting a male and female represent mating. Vertical lines extending downward
from a couple represent their children. Subsequent generations are therefore written
underneath the parental generations and the oldest individuals are found at the top of
the pedigree.
If the purpose of a pedigree is to analyze the pattern of inheritance of a particular trait,
it is customary to shade in the symbol of all individuals that possess this trait.
In the pedigree above, the grandparents had two children, a son and a daughter. The
son had the trait in question. One of his four children also had the trait.
Background
A pedigree is a family tree showing a line of descent. It can be used to trace the
occurrence of inherited traits in parents and offspring through a number of generations.
By convention, circles represent females and squares, males. A line between a square
and a circle represents a union and a line down indicates offspring from the union. Filled
in symbols represent individuals displaying the phenotype being studied. For example:

In pattern 1, the son and father are both affected. This is a reasonable indication that
the characteristic is dominant. An affected offspring must have at least one affected
58

parent if the phenotype is dominant. Other features of pedigrees of a dominant trait


are:
Heterozygous individuals will be affected
Two affected parents can produce an unaffected child (both parents would be
heterozygous)

In pattern 2, the daughter is affected but neither parent is. This can only happen if the
characteristic is recessive and the offspring are homozygous, e.g. bb. Both parent must
be heterozygous, Bb. Other features of pedigrees of a recessive trait are:
Heterozygous parents will be unaffected.
Two affected parents will always have an affected child.
Pedigrees are valuable tools in genetic counseling. It allows a pattern of inheritance to
be traced throughout generations of a family. This can allow identification of the genetic
disease and advice can be made available on the probability of a couple having an
affected child. Cystic fibrosis is an example of a recessive genetic disease. Huntington's
chorea is an example of a dominant genetic disease.
59
60

9. SYNDROMES
Aneuploidy
Aneuploidy is an abnormal chromosomal condition. This is occurred due to non-dis-
junction in autosomes or allosomes. This may either result in loss or gain of
chromosomes in the gamete. Due to this, instead of 2N condition in the zygote, either
2n + 1 or 2n - 1 condition is formed. This addition (gain) 0r deletion (loss) of
chromosome is called aneuploidy. 2n + 1 condition is referred as trisomy or
hyperaneuploidy, by addition of chromosome(s). 2n - 1 condition is referred as
monosomy or hypoaneuploidy, by loss of chromosome(s).

Trisomy [2n + 1]
At fertilization the egg (23 chromosomes) and the sperm (23 chromosomes) fuse to
create a conception, or zygote, which has 46 chromosomes. If a sperm or egg carries an
extra copy of one of the chromosomes, due to non-disjunction at meiosis I or meiosis II,
there will be a total of 24 chromosomes instead of 23 in the reproductive cell. If this
sperm or egg is fertilized by a normal sperm or egg the result will be a total of 47
chromosomes instead of 46.
61

Monosomy [2n – 1]
If one of the gametes has lost any one chromosome, and is fertilized by a normal
gamete the result is monosomy, for the chromosome involved. Monosomy is a
deficiency in number of chromosomes and is defined as only one copy of a chromosome
that is normally present in two copies. These eggs and sperm, which contain one less
chromosome, have 22 chromosomes. When fertilized, the outcome is 45 chromosomes
in total. In general, monosomies are less likely to survive when compared to trisomies.
Syndromes
Syndromes are chromosomal disorders. This is formed due to non-dis-junction either in
autosomes or allosomes. This is causing genetical disorders. If non-dis-junction occurs in
the autosomes, it brings autosomal syndromes. Otherwise if non-dis-junction occurs in
the allosomes, it brings allosomal or sex chromosomal syndromes. Hence the syndromes
are classified into as (i) allosomal or sex chromosomal syndromes and (ii). autosomal
chromosomal syndromes.
62

Klinefelter’s syndrome [2n + 1] 47 XXY


Klinefelter’s syndrome has [2n + 1] condition with 47 XXY chromosomes. This has
occurred due to ND in XX pair of chromosomes. So, one of the gametes is having 22A +
XX chromosomes. On fertilization by a normal gamete having 22A+Y chromosomes, the
zygote will be receiving 22AA+XXY condition. XXY occurs in approximately 1 out of 1,000
live male births, but many men with it do not develop KS. When KS does develop, it
usually goes undetected until puberty or sometimes much later.
Symptoms may include:
For babies:
• Smaller birth weight and slower muscle and motor development
For children and adults:
• Tallness with extra long arms and legs
• Abnormal body proportions (long legs, short trunk)
• Enlarged breasts (common)
• Lack of facial and body hair
• Small firm testes, small penis
• Lack of ability to produce sperm (common)
• Diminished sex drive, sexual dysfunction
• Social and learning disabilities (common)
• Personality impairment
• Normal to borderline IQ
• Speech and language problems—Children with KS often learn to speak later
than other children. They may have a difficult time reading and writing.
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Karyotypes of Klinefelter’s Syndrome and Turner’s Syndrome

Turner’s Syndrome [2n - 1] 47 X


Turner Syndrome is a chromosomal condition first described by Dr. Henry Turner in the
1930’s. Turner’s Syndrome has [2n - 1] condition with 47 X chromosomes. This has
occurred due to ND in XX pair of chromosomes. So, one of the gametes is having 22A + 0
chromosomes. On fertilzation by a normal gamete having 22A+X chromosomes, the
zygote will be receiving 22AA+X condition. It affects only females, and is characterized
by short stature and the lack of normal sexual development at puberty. Turner
Syndrome (TS) affects one in 2,500 live female births and may account for up to 10% of
all miscarriages.
Signs and Symptoms
• A webbed neck (extra folds of skin around the neck)
• Facial features such as a narrow, high-arched palate, receding lower jaw; slightly
drooping eyes, low-set ears, or low hairline
• Scoliosis (curvature of the spine)
• Strabismus (lazy eye)
• Elbows that turn slightly out to the side
• Short fourth metacarpals (the ends of the bones that form the knuckles),
• Flat feet, or small, narrow fingernails and toenails that turn up
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• Female infants with this condition are shorter than average at birth, and may
have fluid around the neck (cystic hydromel) and swelling of the hands and
feet. A broad chest with widely spaced nipples, or specific heart abnormalities
may indicate the presence of Turner Syndrome at birth.
• The intelligence is usually not affected. Sometimes the girl child with TS may
have difficulty with spatial-temporal processing (imaging objects in relation to
one another), non- verbal memory and attention. This may cause problems
with their sense of direction, manual dexterity, and social skills. They may
struggle with maths; however, their verbal and reading skills are often quite
excellent.
• The girl may grow at a normal rate until about three years old, then growth
slows. She will not have the expected growth spurt at puberty. The ovaries will
not produce estrogen and develop eggs. The girl will not begin her menstrual
periods or develop breasts.
• Females with Turner Syndrome often exhibit other physical characteristics
which are associated with TS, and which may vary in severity between
individuals.
Down’s Syndrome [2n + 1] 47 [21st trisomy]
Down’s syndrome has [2n + 1] condition with 47 [21st trisomy] chromosomes. This has
occurred due to ND in 21st pair of chromosomes. So, one of the gametes is having 23A
+ X chromosomes. On fertilzation by a normal gamete having 22A+Y (or)
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22A+X chromosomes, the zygote will be receiving 47 chromosomes, with an additional


chromosome for the 21st pair.
Down’s syndrome Symptoms
• Hypotonia(low muscle tone)
• Palmer Crease
• Nasal Congestion
• Respiratory Infections-bronchitis, pneumonia
• Gastroesophageal Reflux
• Hearing Problems
• Hearing Loss
• Mental Retardation
• Gum disease
• Teeth Problems
• Leukemia
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• Weight Problems – obesity


• Vision Problems – Crossed eyes, Astigmatism, Nearsightedness, Fairsigntedness

Physical Feautres
• Broader nose, Flattened nasal bridge
• Upward slanting eyes, small folds of skin at inner corners
• Small mouth, shallow roof of mouth (makes the tongue look larger when
actually its typical size)
• Teeth – Late eruption, misshapen, missing out of place
• Small ears with the top folded over, small ear passages
• Smaller than normal head (not usually noticeable unless measured)
• Back of the head flattened
• Soft spots on head bigger-take longer to close
• Hands smaller, fingers shorter
• Feet Smaller
• “Sandal Gap” between big toe and rest of toes
• Hair that is soft and thin
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• Sensitive skin, can be mottled and fair


• Shape of chest can be funnel shaped or pigeon shaped
Most kids have only a few Down syndrome symptoms. And symptoms will vary from
child to child.
• congenital heart defect
• mild to moderate low muscle tone
• sandal gap
• one ear is "primitive" (doctor's words)
• sensitive skin, mottled at times (but she is fair-skinned anyway)
• upward-slanting eyes
• flattened bridge of nose and face (this makes her look like a doll. Adorable!)
• some teeth are slightly pointed
• slightly developmentally delayed.
68
69

10. BLOOD GROUPS, ANTIGEN-ANTIBODY REACTION


Blood
Blood is a specialized biological fluid. It is circulating around the body through the blood
vessels. It is considered as a connective tissue
Components of Human Blood
1. Plasma =55%
2. Blood Cells =45%
3. Blood Platelets = 0.1%
Blood group
The different blood types A, B, AB and O provides the best example for the existence of
series of multiple alleles in Man. The observation of agglutination or clumping of the red
blood corpuscles by K Landsteiner (1900-1901) lead to the findings of different blood
groups. Landsteiner found A, B and O blood types. Later, his students Von Decastelo
and Sturli (1902) dicovered AB blood type.
ANTIGEN – ANTIBODY REACTION
Antigen or agglutinogen [a specific protein] present in the red blood cells was found to
react with anti-body or agglutinin [another specific protein] leading to agglutination or
clumping of red blood cells. Two antigens A and B with corresponding two antibodies,
anti-A and anti-B were found. Agglutination observed when the same type of antigen &
antibody were allowed to react. It was observed no individual’s blood had the same
type of antigen and anti – body. Some people were found to have A antigen, some had
B antigen and some had both A and B antigens and some had neither A nor B antigens.
Based on the type of antigen in the RBC, the blood has been typed as A, B, AB and O
respectively. It was also found that A type blood carried B antibody, B type carried A
antibody, AB type with no antibodies and O type carried both A and B antibodies. For
this reason, the clumping of corpuscles and ill effects were reported in the non- related
blood type transfusion.
Agglutination
Agglutination means clumping together of blood cells in the presence of an antibody. It
is an allergic reaction to prevent the foreign materials from the environment or from
others passing inside the individuals
70
71

Rh System
The Rh system was discovered by Landsteiner and A.S.Wiener (1940) from rabbits
immunized with the blood of a monkey, rhesus. The symbol ‘Rh’ is after the species
name of the monkey. Initially the genetic mechanism of Rh System was seemed to be
simple with a single pair of genes R and r, to designate Rh positive (RR/Rr) negative (rr).
Further studies of Niener and R.A.Fisher e+ al revealed the existence of series of
multiple allies, viz R1, R2,R0, Rz,r’,r’’ and ry.
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In general, Rh-positive gene R is dominant over the Rh-negative gene r. These alleles
control the antigen - agglutinogen characteristics of RBCs. The main difference of ABO
system with Rh system is the existence natural antibodies A and B in the former,
whereas there is no such natural antibody in the later. The Rh positive transfusion can
immunize the Rh negative to produce positive antibodies(Rho), but not seen in vice
versa transfusion
Rh incompatibility
Blood transfusion generally occurs between mother and child during pregnancy or
during parturition. An Rh positive(R-) father and an Rh negative (rr) mother may have an
Rh positive child. Always blood transfusion is possible between this mother and child,
and hence mother is possibly immunized to produce Rh posilive(Rho) antibodies. The
first child of this parent, with this genetic background, is nearly always normal.
Ordinarily, atleast one pregnancy (in some cases several) is needed to immunize (or)
sensitize the mother. Thus the subsequent child of this parent would be still born and
showed symptoms of Rh incompatibility. This is clinically termed as Hemolytic disease of
New born (or) Erythroblastosis foetalis. The symptoms with anaemia, janudice and fluid
accumulation in the tissues of the embryo, leading to still born babies (or) infant death
is termed as Rh incompatibility. The genetic combination of Rh positive father and Rh
negative mother only associate with this ilI effects of Rh incompatibility.
To prevent this, the mother is injected with anti-Rh antibodies around the seventh
month of pregnancy and again just after delivering an Rh-positive baby.
Sometime the parents (Rh positive male & Rh negative female) with only one or two
children may never know of their Rh incompatibility, because of the requirement of
higher dose Rh positive blood to immunize the Rh negative mother.
In general before blood transfusion, the blood grouping under ABO system is normally
done. But it is also necessary to know the Rh type of the blood for transfusion. Rh
negative individuals must always be transfused with Rh negative blood to avoid
immunization and subsequent reactions. If the Rh negative individual is wrongly
transfused with Rh positive blood, the Rh positive (Rho) antibodies will be produced in
the Rh negative individual. Again if that individual is transfused with Rh positive blood,
then agglutination is occured by Rho antibodies resulting in the death of the recipient.
Hence, the Rh negative individual must not be given blood transfusion with Rh positive
type, to avoid the Rh incompatibility at a later stage.
73

11. Blood coagulation


Coagulation is a complex process to form a blood clot. It is an important part of
hemostasis.[Hemostasis means the stoppage of blood loss from a damaged vessel]. The
damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding.
This helps to begin repair of the damaged vessel. Any disorder in coagulation may lead
to an increased risk of bleeding (hemorrhage) or clotting (thrombosis).
Coagulation begins almost instantly after an injury to the blood vessel. This injury will
damage the endothelium (lining of the blood vessel). This releases a phospholipid component
called tissue factor and fibrinogen that initiate a chain reaction. Platelets immediately form a
plug at the site of injury; this is called primary hemostasis. Secondary hemostasis occurs
simultaneously: Proteins in the blood plasma, called coagulation factors or clotting factors,
respond through a complex process to form fibrin strands. This strengthens the platelet
plug – the clot.
Mechanism of Blood coagulation
The blood clotting system or coagulation is a proteolytic process. Each enzyme of the
pathway is present in the plasma as a zymogen [ an inactive form ]. On activation, it
undergoes proteolytic cleavage to release an active factor from the precursor molecule.
The coagulation pathway has positive and negative feedback action to control the
activation process. The ultimate goal of the pathway is to produce thrombin. Thrombin
converts soluble fibrinogen into fibrin. Fibrin forms a clot. The generation of thrombin
can be divided into three phases,
1. intrinsic pathways [ Contact activation pathway ],
2. extrinsic pathways [ Tissue activation pathway ],Both provide alternative
ways for the generation of factor X, and.
3. the final common pathway, results in thrombin formation.
Intrinsic pathway
• The intrinsic pathway is activated when the blood comes into contact with the
surface that are exposed as a result of tissue damage.
• This pathway is slower to form fibrin.
• The Hageman factor (factor XII), factor XI, prekallikrein, and high molecular
weight kininogen (HMWK) are involved in this pathway of activation.
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• The first step is the binding of Hageman factor to the surface exposed by an
injury. Thus the factor XII is activated.
• A complex of prekallikrein and HMWK also interacts with the exposed surface
along with the factor XII. During activation, the single chain protein of the native
Hageman factor is cleaved into two chains.
• The light chain contains the active site.
• This molecule is referred to as activated Hageman factor (factor XIIa).
• Activated Hageman factor in turn activates prekallikrein.
• The kallikrein cleaves further the factor XII. This triggers the clotting
mechanism.
• The activated factor XII activates factor XI.
• This next step requires Ca++ to proceed efficiently.
• The HMWK [high molecular weight kininogen] also binds to factor XI to facilitate
the activation process.
• Eventually the intrinsic pathway activates factor X.
• Factor X is the first molecule of the common pathway and is activated by a
complex of molecules containing activated factor IX, factor VIII, calcium, and
phospholipid. This is provided by the platelet surface.
• The precise role of factor VIII in this reaction is not clearly understood. Anyhow,
its presence in the complex is obviously essential.
Extrinsic pathway
• The extrinsic pathway is an alternative way for the activation of the clotting
process. The main role of this pathway is to generate a "thrombin burst".
• It provides a very rapid response to tissue injury.
• It generates the activated factor X almost instantaneously, compared to the
intrinsic pathway to activate factor X.
• The main function of the extrinsic pathway is to increaset the activity of the
intrinsic pathway.
• There are two components unique to the extrinsic pathway, tissue factor or
factor III, and factor VII.
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• Tissue factor is present in most human cells bound to the cell membrane. The
activation process for tissue factor is not entirely clear.
• On activation, the tissue factor binds rapidly to factor VII. Then factor VII is
activated.
• This form a complex of tissue factor, activated factor VII, calcium, and a
phospholipid.
• This complex then rapidly activates factor X.
COMMON PATHWAY
• The intrinsic and extrinsic systems converge at factor X to enter into this
common pathway
• This pathway is ultimately responsible for the production of thrombin (factor
IIa).
CLOT FORMATION
• The end result of the common pathway is the formation of thrombin.
• It helps the conversion of fibrinogen to fibrin.
• Fibrinogen is a dimer soluble present in the plasma.
• Exposure of fibrinogen to thrombin leads to rapid proteolysis of fibrinogen. It
releases fibrinopeptide A.
• Further proteolysis of fibrinogen second peptide, fibrinopeptide B.
• The fibrin monomers formed by this second proteolytic cleavage polymerize
spontaneously to form an insoluble fibrin gel.
• The polymerized fibrin is stabilized by the transamidating enzyme factor XIIIa.
• This insoluble fibrin aggregates (clots) together with aggregated platelets
(thrombi).
• This clot blocks the damaged blood vessel and prevent further bleeding.
• This is called blood coagulation.
• A thrombus or blood clot, is the final product of the blood coagulation step in
hemostasis.
• Hemostasis means the cessation of blood loss from a damaged vessel.
• It is achieved by the aggregation of platelets that form a platelet plug, and
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• the activation of the humoral coagulation system (i.e. clotting factors).

• A thrombus is normal in cases of injury.


1. Bleeding Time : It is referred as the time taken for small blood
vessels to close.
2. Clotting Time : It is referred as the time taken for fibrin clots to
form
Eventually, blood clots are reorganised and resorbed by a process termed fibrinolysis.
The main enzyme responsible for this process is plasmin. It is regulated by various
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activators and inhibitors. Thus coagulation is a complex process wherein blood forms
solid clots by fibrin-containing platelets.
Factors Affecting Coagulation
• amount of calcium and phospholipids in the body
• amount of Vitamin K
• Presence of protein C, anathothrombin & TFPI (Tissue factor pathway inhibitor)
Blood sugar
Blood sugar concentration, or glucose level, is regulated by the metabolic homeostasis.
It is meaning to the amount of glucose present in the blood of a human. Normal blood
glucose level in man is about 90 mg/dl. The total amount of glucose normally in
circulating human blood is therefore about 3.3 to 7g (assuming an ordinary adult blood
volume of 5 litres). Glucose level raises after meals for an hour or two by a few grams. It
is usually lowest in the morning, before the first meal of the day. Sugar is transported
through the bloodstream from the intestine, or liver to body cells. Glucose is the
primary source of energy for body's cells.
Any failure in the regulation to maintain blood glucose shall result either in low level of
sugar in the blood [Hypoglycemia] or in high level of sugar in the blood [Hyperglycemia].
Diabetes mellitus is characterized by the hyperglycemia. It is the most prominent
disease related to failure of blood sugar regulation.
Normal values of blood sugar
Blood glucose level normally remains within a remarkably narrow range. In most
humans this varies from about 82 mg/dl to perhaps 110 mg/dl. But shortly after eating,
the blood glucose level raises temporarily up to 140 mg/dl or a bit more in non-
diabetics. The diabetes association recommends a post-meal glucose level less than
180 mg/dl and a pre-meal plasma glucose of 90-130 mg/dl.
Blood sugar regulation
The homeostatic mechanism is keeping the blood glucose level in a remarkably narrow
range. It is composed of several interacting systems. It is done by hormones. There are
two types of mutually antagonistic metabolic hormones regulating the blood glucose
levels.
• catabolic hormones (such as glucagon, growth hormone, cortisol and
catecholamines). They increase blood glucose level; and
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• anabolic hormone (insulin), It decreases blood glucose level.


Some edible mushrooms are well known for the ability to lower blood sugar levels
including Reishi, Maitake, Agaricus blazei as well as some others.

Blood sugar levels are regulated by negative feedback in order to keep the body in
homeostasis. The levels of glucose in the blood are monitored by the cells in the
pancreas's Islets of Langerhans. If the blood glucose level falls to dangerous levels (as in
very heavy exercise or lack of food for extended periods), the alpha cells of the pancreas
release glucagon. This hormone helps to increase blood glucose levels. It converts
glycogen into glucose. (This process is called glycogenolysis). The glucose is released into
the bloodstream, increasing blood sugar levels.There are also several other causes for
an increase in blood sugar levels. Among them are the 'stress' hormones such as
adrenaline, several of the steroids, infections, trauma, and of course, the ingestion of
food.
When levels of blood sugar rise, a different hormone is released from beta cells found
in the Islets of Langerhans. This hormone, insulin, causes the liver to convert more
glucose into glycogen. (this process is called glycogenesis). Also forces about 2/3 of body
cells (primarily muscle and fat tissue cells) to take up glucose from the blood. Thus the
blood sugar level is decreased
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Complications
Diabetes mellitus type 1 is caused by insufficient of insulin. Diabetes mellitus type 2 is
primarily due to a decreased response to insulin in the tissues of the body (insulin
resistance). Both types of diabetes, if untreated, result in too much glucose remaining in
the blood (hyperglycemia). Both have many of the same complications. Also, too much
insulin and/or exercise without enough corresponding food intake in diabetics can result
in low blood sugar (hypoglycemia).
Health effects
Dropping of blood sugar level leads to a potentially fatal condition called hypoglycemia.
Symptoms include lethargy, impaired mental functioning, irritability, shaking, weakness
in arm and leg muscles, sweating and loss of consciousness. Brain damage is even
possible.
If blood sugar levels raises too high, appetite is suppressed over the short term. Long-
term hyperglycemia causes many of the long-term health problems associated with
diabetes, including eye, kidney, heart disease and nerve damage.
Blood Cholesterol (“chole”-means bile & “stereo” means solid)
Cholesterol is essential to life. But too much of the wrong kind of cholesterol increases
the risks of heart disease. It is a compound – a steroid – that occurs naturally in the
body. It is manufactured by the liver. It is essential for many of the body's metabolic
processes. It helps make hormones like oestrogen, testosterone and adrenaline. It is
used in the production of vitamin D. Also in the production of bile acids, which help the
body to digest fat and absorb fat-soluble vitamins in the small intestine.
The trouble starts with the increase of too much cholesterol level in the blood. The
intake of fats in our diet causes the levels of cholesterol in our blood to rise to more
than the optimum. The optimum level is 5.5 millimoles per litre. If it rises above the
normal level, it is added as fatty deposits on the surface of our arteries. These narrow
the arteries and block blood from flowing, leading to heart disease, stroke and other
conditions. This is a condition known as atherosclerosis. High cholesterol is one of the
risk factors for atherosclerosis, along with smoking, being overweight, and having high
blood pressure.
Good fats, bad fats
Most cholesterol is manufactured in the liver from fats in our diet. Cholesterol is
attached to carrier molecules made of fat and protein called lipoproteins.
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There are two major types of these 'carrier' lipoproteins – low-density lipoprotein (LDL)
and high-density lipoprotein (HDL). LDL is the major carrier of cholesterol from the liver
to the rest of the body. The increased cholesterol level induces the LDL to deposit the
cholesterol onto the surfaces of the arteries. This causes the damage to the artery. But
the HDL clears-up cholesterol from the bloodstream. HDL takes the cholesterol back to
the liver. So it reduces cholesterol level in the blood. Thus it reduces the chance of it
being deposited in the arteries.
HDL cholesterol is sometimes called 'good cholesterol' and LDL cholesterol 'bad
cholesterol'. The higher the ratio of HDL to LDL – the lower the risk of artery disease.
Normal levels of cholesterol
• Total cholesterol: Less than 5.5 mmol/l
• LDL: Less than 3.5 mmol/l
• HDL: Greater than 1.0 mmol/l
• LDL to HDL ratio: Less than 4
• Triglycerides: Less than 2.0 mmol/l.

It's worth taking steps to lower cholesterol. Because lowering the cholesterol by 10 per
cent reduces the risk of heart attack by 20 per cent. Regular exercise[ at least 30
minutes of brisk walking daily ] increases HDL levels and reduces LDL levels in the body.
81

12. REPRODUCTIVE CYCLE IN MAN


MENSTRUAL CYCLE
Each month women of reproductive age has a cycle of fertility. This results in either
pregnancy or menstruation. The average menstrual cycle is 28 days but ranges from 24
– 35 days. The monthly recurrence of this event, together with other regular body
changes, constitute the female reproductive cycle. Its most obvious external sign is
menstruation (monthly bleeding). For this reason, the reproductive cycle may also be
called menstrual cycle. The menstrual cycle happens roughly every 28 days during a
woman’s fertile or reproductive years. In this cycle, the ovum (egg) travels from the
ovary after maturing to the womb (uterus). If the ovum is not fertilized, then pregnancy
will not occur. If the pregnancy is not happened, the lining of the uterus[endometrial
wall] is sloughing or shedding off. Then it is passed out as a bloody discharge. The
average blood loss during menstruation is 35 ml [ 10–80 ml considered normal]. This
discharge of blood is known as a “woman’s period”. Menstruation is also called
menstrual bleeding, menses, catamenia or a period.
First and last menstruation
A girl's first menstruation [menarche] usually occurs between the ages of 11 and 13.
During adolescence, menstrual cycles are rather irregular. It is only later that some
definite pattern is established. In a mature woman, menstrual cycles usually last
between 28 and 35 days. Still, some irregularity is always possible and quite normal. The
irregularity increases again as the woman grows older. The cessation of menstrual cycles
at the end of a woman's reproductive period is termed menopause.
REPRODUCTIVE HORMONES
The menstrual cycle is under the control of the endocrine system. The hoemones are
necessary for reproduction.
LUTEINIZING HORMONE
Leuteinizing hormone or LH is produced by the pituitary gland. This hormone is released
to make the ovary to release the mature egg.
FOLLICLE STIMULATING HORMONE
FSH is produced by the pituitary gland. FSH stimulates the ovaries for maturing the
egg(s) and into producing estrogen.
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ESTROGEN
Estrogen is produced by the ovaries. Estrogen causes the cervical fluid to be thin , and
abundant and causes the basal body temperature to be low.
PROGESTERONE
Progesterone is produced by the corpus luteum . Progesterone is the hormone released
after ovulation. Progesterone is responsible for the basal body temperature rising and
for maintaining the uterine lining – in preparation to receive the fertilized egg.
HUMAN CHORIONIC GONADOTROPHIN
HCG is referred to as the pregnancy hormone. HCG is produced by the developing
embryo when it implants into the uterine lining. HCG helps to maintain the corpus
luteum in the event of pregnancy which maintains the high levels of progesterone.
Menstrual cycle
The menstrual cycle is divided into several phases. The average length of each phase is
shown below:

MENSTRUAL PHASE (1 - 5 DAYS )


The first day of women’s period is considered the first day of the menstrual cycle . The
unfertilized egg disintegrates. If the pregnancy is not happened, the lining of the
uterus[endometrial wall] is sloughing or shedding off. Then it is passed out as a bloody
discharge. Low levels of hormones estrogen and progesterone during this phase cause
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the endometrium to break down and be shed in the form of menstrual blood. Bleeding
is lasting on an average of 5 days. Cramping in the abdomen, back, or upper thighs are
common symptoms during the first few days of menstrual phase..
FOLLICULAR PHASE (5 – 13th DAY)
The follicular phase (or proliferative phase) is the phase of the estrous cycle. It ends
with ovulation. Early in the menstrual cycle the pituitary gland in the brain produces
rising amount of follicle stimulating hormone (FSH). It acts on ovaries to promote the
development of several follicles. Each follicle is containing an egg. Only one follicle will
attain maturity. Towards the end of this phase, the ovaries secret increasing amount of
estrogen, which causes the uterine lining to begin thickening in preparation to receive
the fertilized egg.
OVULATORY PHASE (around 14th day )
The pituitary gland and hypothalamus releases a surge of lutenizing hormone (LH) about
midway through the cycle. This causes the mature follicle to bulge out from the surface
of the ovary and burst, releasing the egg. Ovulation usually occurs around day 14 of the
cycle .The egg then begins to travel down the fallopian tube and into the uterus. This is
the time when women are most likely to become pregnant. In some women, ovulation
features a characteristic pain called mittelschmerz (German term) meaning middle pain.
The sudden change in hormones at the time of ovulation sometimes also causes light
mid-cycle blood flow.
Signs of Ovulation
There are some common physical symptoms of ovulation that may be observed.
1. increased body temperature
2. increased cervical mucus
3. abdominal cramps (Mittelschmerz)
4. tender breasts
LUTEAL PHASE (15 – 28th DAY)
The luteal phase is also called the secretory phase. An important role is played by the
corpus luteum. After releasing the egg the ruptured egg develops into a structure called
corpus luteum. It secrets increasing amount of progesterone. Progesterone plays a vital
role in making the endometrium receptive to implantation of the blastocyst. It is
supportive of the early pregnancy.
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If the egg is fertilized the corpus luteum begins to produce HUMAN CHORIONIC
GONADOTROPHIN (the hormone that pregnancy test detects), which maintains the
corpus luteum and its progesterone secretion. The egg moves to the uterus and
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attaches to the endometrium about 6-7 days after ovulation, where it begins to develop
into a fetus.
If the egg is not fertilized, the corpus luteum degenerates after about 14 days and the
level of progesterone and estrogen drop. This causes the endometrium to break down
and shed and a new menstrual cycle begins.
Fertile window
The most fertile period is the days with the highest likelihood of pregnancy. It is some 3
days before and until 1–2 days after ovulation., ie., 11th to 16th day of the cycle. In an
average 28 day cycle with a 14-day luteal phase, this corresponds to the second and the
beginning of the third week. By knowing the cycle period, estimation of the relatively
fertile and the relatively infertile days in the cycle can be understood. These systems are
called fertility awareness.
Birth control
Birth control or contraception is a way to allow a man and woman to reduce the chance
of pregnancy while having sexual intercourse. It is called safe sex. Some of the
contraceptives available can also protect a person from sexually transmitted diseases.
Birth control is also sometimes called family planning. This is because it means that
people have babies and make their families when they plan for it.
Birth control may be used by married couples, or by couples who live together but are
not marrried, or by a man and woman who are engaged, or by a couple who are merely
boyfriend and girlfriend, or even by single people who are not in a relationship at all but
who may have casual sex with others.
Types of birth control
There are many types of birth control. Some of these have been done for a long time.
Many of them were only discovered in the last eighty years. Each type of birth control
has advantages and disadvantages.
1. Barrier methods
2. Hormonal methods
3. Intrauterine methods
4. Sterilization
5. Traditional contraception
6. Rhythm method
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7. Induced abortion
1. Barrier methods
A barrier is something that stands between two things. So barrier methods stand
between the sperm and the ovum (egg). Some barrier methods help prevent many
sexually transmitted diseases (STDs).
Condoms
• Male Condom – This is the oldest barrier method. A condom is a thin tube
(often made of latex or plastic) that the man puts over his penis. This keeps the
sperm from getting to the egg. Condoms are low-cost and easily available. They
also are the only form of birth control that will protect both partners against
most sexually transmitted diseases.
Failure rate: About 15 out of every 100 couples each year may become pregnant
using condoms.
• Female condom – This is like a male condom. It goes into the woman's vagina.
Failure rate: About 21 out of every 100 couples each year may become pregnant
using female condoms.
• Diaphragm and cervical cap – These are objects that a woman puts in her vagina
to cover the cervix. Diaphragm: Made of latex and shaped like a dome with a
flexible rim, the diaphragm is inserted into the vagina prior to sex to cover the
opening of the cervix.
Failure rate: About 16 out of 100 couples.
• Cervical cap: This sailor's hat-shaped silicone cup is inserted into the vagina and
over the cervix.
Failure rate: About 29 out of 100 couples. [ Less effective for women who have
previously given birth than for women who have not ].
• Contraceptive sponge – This is a plastic foam sponge that is filled with
spermicide (a substance that kills sperm). It is inserted in the woman's vagina
over the cervix.
Failure rate: About 16 to 30 out of 100 couples [ Less effective for women who
have previously given birth than for women who have not ].
Barrier methods can be easy to use and have few side effects (bad things that happen if
you take a medicine). Some of them can be bought without a doctor's prescription. But
sometimes they can be messy or interfere with the pleasure and sensation of sex.
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2. Hormonal methods
These can only be used by women. Investigations are going on to find a way to use
hormonal methods for men. Hormonal birth control is extremely effective, if it is used in
the right way. Many hormonal birth control methods also make women's menses
shorter and with less bleeding, which most women like. It does not interfere with sex.
For all of these reasons, hormonal methods are very popular.
But all hormonal birth control methods have some risks for side effects. They can make
a very small increase in the risk of blood clots in the lungs, strokes, heart attacks, and
breast cancer. But these risks are not as big as the risk a woman has if she gets
pregnant.
Also, hormonal methods do not prevent STDs.
Birth Control Pills
• Birth Control Pills – Birth control pills are made of hormones -estrogen and
progesterone. This prevents a woman’s ovaries from releasing an egg each
month. Thus it stops ovulation. If there's no egg to join with a sperm, no
pregnancy will occur. The hormones also thicken a woman's cervical mucus. It
also helps to slow down and block sperm headed for the uterus. The pill has to
be taken every day, whether or not the woman has sex, to be effective as birth
control.
Failure rate: Fewer than 1 out of every 100 couples may become pregnant if the
pill is taken perfectly - every day, at about the same time each day. But about 8
out of 100 couples may become pregnant if the pill isn't taken each day as
directed.
• Birth Control Patch –This is a small and thin object that is put on the woman's
skin and stays there. Hormones in the patch go into the skin and into the
woman's body. This prevents ovulation. It follows the three-weeks on, one-
week off pattern.
Failure rate: About 8 out of 100 couples.
• Emergency contraception pill – also called the day-after pill. This is a medicine
that is taken after sex that makes the woman less likely to get pregnant. It is
best if used very soon after sex. It is recommended to be taken no more than 3
days after the event. After this time, the pill no longer works well.
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• Implants – these are objects that are put under the woman's skin and stay there
for years. They slowly put hormones into her body and make her not ovulate.
These only have progesterone in them (not estrogen).
• Injections (shots) – This is a hormonal injection [ progesterone hormone ] to the
woman. This shot by an injection needle is given for every three months. This
prevents ovulation.
Failure rate: About 3 out of 100 couples
• Vaginal ring - This contraceptive ring is flexible and small and is inserted into the
vagina. The hormones in the ring go into the woman's vagina and into her body
to prevent her from ovulating. The ring cannot be felt during sex. One ring lasts
for up to three months.
Failure rate: About 8 out of 100 couples.
• Implants and injections are very good for younger women or women who
cannot take a pill every day.
3. Intrauterine methods
• IUD, or intrauterine devices: They are small, shaped like the letter “T”. It is made
of flexible plastic. IUDs are the most common birth control method used in the
world. They have many good things about them. It is inexpensive. It lasts for a
long time, even upto 10 years. They are placed by a gynecologist into a woman's
uterus. IUDs prevent sperm from joining with an egg, but it's not entirely clear
how they work. There are some health risks involved. It is often preferred to use
IUDs in women who've already had a baby.
Failure rate: Less than 1 out of 100 couples.
4.Sterilization
This is the ultimate birth control method. Sterilization is extremely effective. In this
method surgeons made pregnancy impossible by permanently altering the reproductive
system. Generally speaking, these procedures are very hard to reverse.
• Vasectomy:. This is a small surgery done in the tubes that carry sperms. The
tubes are closed or blocked to transmit sperm into the seminal fluid released
during ejaculation. The blocked sperm are absorbed by the man’s body. The
ejaculate released during sex is incapable of causing pregnancy.
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• Tubectomy:. In this surgery, the fallopian tubes are cut or clipped. So that the
eggs cannot go down them to the uterus. (The fallopian tube is the tube that
carries the egg from the woman's ovary).
5. Traditional contraception
These methods are used for a very long time. They were used before modern medicine.
• Coitus interruptus (sometimes called "withdrawal" or "pulling out"): This is not a
good method of birth control because the fluid that comes out before semen
comes out also has sperm in it. So if the man's semen is close to the woman's
vagina, pregnancy is possible.
• Non-vaginal sex – This is a method of sex without putting the copulatory organ
in the woman's vagina. This is more effective than coitus interruptus. But it is
still risky if the man's semen is close to the woman's vagina, pregnancy is
possible.
• Abstinence – This is a method of not having sex. If a couple can practice this
correctly, it is completely effective at preventing pregnancy. It is free and does
not require the help of a doctor. Many methods can reduce the risk of STIs, but
only abstinence is 100% effective.
• Periodic abstinence -- This method means a man and a woman practice
abstinence (not having sex) when the woman is fertile.
6.Rhythm method
This is where a woman records the number of days between her menses (the time when
she bleeds). There is a calculation that the man and woman may aware of themselves,
when the woman is more fertile and less fertile. For some women, this calculation does
not work.
7.Induced abortion
Induced abortion (sometimes called just abortion) is when a doctor gives a pregnant
woman a medicine or does a surgery to eliminate the pregnancy. Some do not call
abortion a kind of contraception. This is because contraception means preventing
pregnancy, but abortion is stopping a pregnancy that has already started. Abortion is
not a good birth control method.
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91

13. CONSTRUCTION OF rDNA


Introduction
Construction of rDNA is done by recombination DNA technology. Recombinant DNA
Technology is also called Gene Cloning or Genetic Engineering.
Definition
Recombinant DNA Technology is a novel technique for controlled recombination of
DNAs between totally incompatible genomes in a short time giving the cells new and
increased synthetic capabilities.
Making bacteria synthesize human hormones is an example.
Steps Involved
1. Selection of the desired gene (gene of interest/foreign gene/target
DNA/passenger gene) and its isolation.
2. Selection of a suitable, naturally available vehicle or cloning vector to carry the
desired gene (or) the construction of a desired gene in vitro.
3. Selection of suitable enzymes for gene-splicing and ligation (cutting and
rejoining the DNAs).
4. Insertion of desired gene into plasmid.
5. Introduction of the recombinant DNA (rDNA/hybrid DNA/Chimeric DNA), with
an altered gene sequence, into a host cell for replication and producing more
copies.
6. Identification of rDNA inside the host cell.
7. rDNA amplification through Polymerase Chain Reaction (PCR).
8. Maximizing gene expression.
9. Expressed products of the rDNA inside the host cells (recombinants).
1. Selection of Desired Gene
a) From Natural Source
The cells are washed with sodium chloride solution. The cell walls are ruptured using
homogenization. The homogenized cells (homogenates) are treated with sodium
dodecyle sulphate (SDS). Then, they are centrifuged. The proteins and RNA in the
supernatant are removed. DNA alone now remains. Ethanol is added and stirred well
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with a rod. A white fibrous precipitate forms. It is dissolved in ethanol-sodium chloride


solution. It contains DNA. This is the desired gene.
b) From mRNA
1. The cells containing large amounts of the needed mRNA are identified and
isolated.
2. mRNA is then isolated.
3. The enzyme, reverse transcriptase and nucleotides are added.
4. Reverse transcriptase helps in the synthesis of the complementary strand to
the template mRNA.
5. The newly formed single DNA strand is called cDNA or
complementary DNA.
6. The enzyme DNA polymerase and nucleotides are added.
7. DNA polymerase catalyzes the synthesis of a complementary second strand to
the cDNA.
8. This is the desire gene.
2. Selection of a suitable, naturally available vehicle or cloning vector
Plasmid is a circular and double-stranded DNA. It contains (a) an origin of replication,
(b) marker genes and (c) one or more restriction sites. It is found in bacteria. First, the
bacterial cell wall is ruptured using Lysozyme and EDTA. Then, the cell is lysed with
sodium lauryl sarcinate (SLS) and centrifuged. Plasmids separate.
3. Selection of suitable enzymes for gene-splicing and ligation
The splicing enzymes are called restriction enzymes. They function as chemical knives.
They help to cut the plasmid and desired DNAs at certain places called restriction sites
(with specific nucleotide sequence).
The restriction enzymes cut the DNAs in two ways. In one, the cut-ends are unequal in
length. Such ends are called sticky or cohesive ends. In the second, the cut-ends are
equal in length. They are called blunt ends. Ligases are enzymes which unite the
plasmid and the desired DNA.
4. Insertion of desired gene into plasmid
The cut plasmids and the desired genes are mixed together. The desired gene
incorporates into the plasmid. This is called ligation. The plasmid DNA with the desired
gene is called the recombinant DNA. (rDNA / hybrid DNA / Chimeric DNA)
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5. Introduction of the recombinant DNA into a host cell


The rDNA can be introduced into a bacterium, plant cell or animal cell. This receiving
cell is called transformed cell, transformant or recombinant. This process is called
transformation. The introduced new DNA (rDNA) integrates with the host (transformed
cell) DNA. The other methods of introduction of the rDNA into host cells are (a)
Electrporation, (b) Shot-gun method or biolistics, (c) Microinjection, (d) transduction
(e) Transfection and (f) Liposome-mediated fusion.
6. Identification of rDNA inside the host cell
The rDNA can be identified by three methods: insertional inactivation, immunochemical
method and colony hybridization.
7. rDNA amplification through Polymerase Chain Reaction (PCR)
This method enables specific DNA sequences to be selectively amplified a thousand- or
even a million fold.
8. Maximizing gene expression
The purpose of cloning a desired gene is to produce the product of that gene in large
quantities for commercial use. So, gene expression is maximized.
9. Expressed products of the rDNA
Many hormones and enzymes useful to mankind, cattle, fermentation, etc. are
produced. Examples: Insulin, Somatotropin, Human growth hormone, vaccines, Human
interferon, Urokinase, amino acids, single cell proteins (SCP), transgenic plants and
animals, etc.
Advantages
DNA finger-printing, formation of genomic library, etc.
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14. FERMENTATION
Introduction
Fermentation is known to man since a long time. Microbial processes were utilized for
the production of food, medicines and alcoholic beverages. Even before 6000 B.C., the
Sumerians and the Babylonians knew to make alcohol – beer – by yeast. By about 4000
B.C., the Egyptians discovered that the brewer’s yeast produced carbon dioxide. It
leavened the bread. In the Scriptures too, there is mention about wine. The curd, the
Indian ‘idly’, etc., are some of the proofs that the ancients used fermentation.
Actually, microbes can act on any carbon-rich material. Nowadays, carbon dioxide is
directly tried as a raw material.
Definition
Fermentation is a biochemical energy-generation process of transformation by which
microorganisms, either in the absence of oxygen or in the presence of low oxygen, turn
raw materials ( substrates ), which act as both electron donors and electron
acceptors, into more useful products, the (Metabolites).
The Process

3
1 2

The process involves three stages. They are


1. the upstream,
2. the actual process and
3. The downstream.
1) The Upstream
The upstream stage involves many steps.
(I) the Bioreactor or Fermentor or Fermentation Vessel
They are the vessels for microbial cultures under controlled conditions. Micro-
organisms are grown in them. According to requirements, there are different types.
(A) Culture Tube Slant
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This is used in labs. Test tubes are the fermentors. A single microbial culture is grown
and maintained.
(B) Erlenmeyer Flasks
They are simple structures. They are of different volumes, 100-1000 ml.
(C) Baffle Flasks
A dent is made in the side of the flask. Due to this, the inner surface is not continuous
and smooth. This increases turbulence of the culture medium. As a result, oxygen
supply and growth of micro-organisms increase.
(D) Shakers
These vessels are always shaking. The shaking frequency can be adjusted. Microbial
growth increases.
(E) Fermentors or Bioreactors
They are large. They are used for mass production in industries. They are designed to
provide a controlled environment.
(a) Designing and constructing a Bioreactor
1. The bioreactor must be large.
2. It must have the capacity to work continuously for a long time.
3. It must have provision for aeration.
4. It must have a device to shake or agitate the culture.
5. It must have the facility for temperature regulation.
6. It must have the facility for pH regulation.
7. It must utilize only less energy.
8. It must have a sampling device.
9. Medium should not get evaporated.
10. Its handling must be easy.
11. The internal surface must be smooth.
12. It must be reusable.
13. It must be easily cleaned, serviced and maintained.
14. It must work even with cheap raw materials.
15. Their size must be uniform.
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There are different types of fermentors. The most common is the Stirred Tank
Fermentor.
Structure of Stirred Tank Fermentor

Motor
Culture or Nutrient
Addition Stirrer

Sample Line Cooling Water Outlet


pH Probe

Dissolved O2 Probe

Temperature Sensor Impellers


& Control Unit
Cooling Jacket

Cooling Water Inlet Biosensor Unit

Air Filter

Harvest Line

It is a vertical, cylindrical vessel. Its both ends are closed with hemispherical basins. A
stirrer is positioned inside. A heating coil is attached for temperature regulation.
Monitors for pH and dissolved oxygen are inserted. The vessel is surrounded by a water
jacket.
At the top of the vessel, there are two openings. One is for adding the inoculums. The
second is for releasing out the gases produced during fermentation. There are two
openings on the sides. The first is for adding the culture medium. The other is for
collecting the products. Below the stirrer is inserted a pipe. Through it, sterile air is sent
in. The water jacket has two openings. One pumps in cool water. The other, pumps out
the circulated water.
(II) Selective Methods
Microorganisms usually occur together. They have to be separated according to the
needs. By this, pure cultures are got. It increases the number of that species. It is done
by two methods.
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A. Physical Methods
Heat Selection
Through this, endospore forming bacteria are separated. The mixed culture will be
heated at 800 C. for 10 minutes. All vegetative cells die.
pH
pH selects acid-tolerant and other microbes.
Cell Size and Motility
An agar plate is taken. A membrane filter is placed over it. It has a pore size of 0.15µ.
The small sized spirochaetes pass down through the filter. Others do not.
B. Chemical Methods
Use of special Carbon and Nitrogen sources
This method is called enrichment. In a mixed culture, nitrogen gas alone is used as
nitrogen source. Nitrogen-fixing bacteria only will grow.
Use of inhibitory chemicals
Crystal violet dye, bile salts, etc., allow the growth of gram-negative bacteria and inhibit
gram-positive.
III Methods of isolating pure cultures
(A) Streak Plate Technique
Pour Plate and Spread Plate Methods.
IV Preparation of Culture Medium
The culture media provide the optimum growth factors like pH, osmotic pressure,
chemical factors, etc. They are of two types.
(A) Natural Media
They are the natural sources of nutrients. They are enough for necessary growth and
development.
(B) Synthetic Media
They are prepared artificially. For different microbes, different types of synthetic media
are prepared. Several nutrients are added. The common nutrients needed and their
raw materials are as below.
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• Carbon source - Cane molasses, glucose, etc.


• Nitrogen source - Ammonium salts, Soya-bean, meal, yeast extracts,..
• Minerals - Calcium copper, magnesium, manganese, Phosphorus,
potassium, sulpher,zinc, etc…
• Growth Factors - Proteins.
• pH - 6 – 8.
• Buffering agents
V Sterilization of Medium
The medium prepared may contain some unwanted or even harmful microorganisms.
Sterilization deactivates them. Moist- Heat Method is usually used. This is done with
steam pressure. Autoclaving is also employed.
VI Stock-culture Production
The selected strains of microbes are cultured in small flasks. They will be used in the
fermentors. So, they are called stock-cultures. Then, they are subcultured in large-
scales. Now, they are called inoculum. They are directly added to the Fermentor.
VII Culture of Microorganisms in the Bioreactor
First, the bioreactor is sterilized properly. Then, it is washed with sterile water. The
sterilized liquid medium is introduced into it. Then, the inoculum is added. This can be
done in three different ways.
A) Batch Culture
In this method, a desired microbe is grown in a closed culture system on a limited
amount of medium of microbial culture. The microbial growth stops under two
conditions. (1) The nutrient is completely used up. (2) Toxic metabolites are produced
by the microbes. Because of this, used medium is removed and new medium is added.
B) Continuous Culture
In this type, the growth stops when the entire medium is used. So, the used medium is
continuously removed. New medium is added in its place. The microbes grow
continuously.
C) Fed-batch Culture
A certain amount of medium is removed at specific intervals. New medium is added
periodically. Microbes grow continuously.
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3) The Downstream
After the fermentation process is over, the microbial products accumulate in the
medium. They should be separated, purified, processed and packaged. This activity
sequence is called downstream processing.
A) Separation of Biomass
The product may be the biomass itself (the multiplied microbes) or some other desired
thing produced by the microbes. If the biomass itself is the product, it is called Single
Cell Protein (SCP) or vaccines. Some flocculating agents are added. The microbial cells
settle down. The fermented broth (medium + biomass) is centrifuged or ultra-
centrifuged. The used up medium and the biomass separate. The biomass is collected.
Ultra-filtration or continuous filtration can also be used.
B) Cell Disruption
The desired product may the intracellular vitamins, enzymes, human insulin, etc. To get
them, the microbial cells must be broken. These products will be released. By
centrifugation or filtration, they are separated.
C) Concentration of Broth
Sometimes, the product may be secreted by the microbes. They become extra-cellular.
Then, the used up medium is concentrated. The product is separated.
D) Initial Purification of Metabolites
The different products vary physico-chemically. So, the extraction methods are also
different. They may be precipitation, solvent-extraction, ultra-filtration,
chromatography, etc.
E) Purification
Extra quantity of water is removed. This is called de-watering. Then, the product is
purified to nearly 100%. It is packaged and marketed.
FERMENTATION PRODUCTS
Alcohol, Antibiotics, Beverages, Enzymes, Organic Acids, Polysaccharides, etc.
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15. ENZYME TECHNOLOGY


Definition of Enzymes
Enzymes are biocatalysts which accelerate biological reactions.
Enzymes – A General Account
Enzymes are produced by plants, animals and microorganisms. Many of them are used
in food industries, leather softening, manufacture of detergents, etc. Microbial
enzymes have become more popular now. They help in the production of primary and
secondary metabolites by the microorganisms.
Microbial Enzymes
There are two types of microbial enzymes. They are (1) extra-cellular and (2)
intracellular.
The extra-cellular enzymes are secreted outside the microbial cell. Those produced by
the saprophytic and pathogenic microorganisms are important. Cellulase,
polymethylgalacturanose, polyglacturonase, pectinmethyesterase, etc. are some of
them. They help in the establishment of the host tissues or decomposition of organic
substrates.
The intracellular enzymes are secreted inside the cell. Example: Invertase, uric oxidase,
asparaginase, etc. They are economically important. They cannot be easily extracted
from inside the microbial cell.
Microbial enzymes are more advantageous than the animal and plant enzymes.
• Production depends on the fermentor size, type of microbial strain and growth
conditions.
• They are economical, can be produced in large scale, limited time and space.
• They are easily extracted and purified.
• Microbes produce a wide variety of enzymes.
• They can grow in any environmental conditions.
• They can be genetically altered easily.
• This increases enzyme production.
• Their life cycle is short.
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At present, more than 2000 enzymes have been isolated and studied. 1000 of them are
used in many applications. 50 of them are industrially used.
Microorganisms Producing Enzymes
Bacteria, Actinomycetes and fungi produce many enzymes.
Properties of Enzymes
Species Specificity
Macromolecules like proteins are species specific. This variation is the reason for the
phylogenetic variations of the microbes. The same type of enzymes may be present in
different microbe species. But, each enzyme acts differently.
Variation in Activity and Stability
The stability and activity of the extra-cellular enzymes are controlled by the optimum
environmental conditions for microbial growth. The environmental factors are pH,
temperature, enzyme concentration, substrate concentration, presence of ions, reduced
amount of water content in the reaction mixture, etc. Each factor influences the activity
and stability of the same or different enzymes in different ways.
But, the intracellular enzymes are not affected by the environmental factors.
Substrate Specificity
Organic matter is complex. It has many substrates like cellulose, hemi-cellulose, lignin,
etc. All microbes cannot decompose all of them. One microbial population may
decompose cellulose. After that, its place may be taken by the lignin – decomposing
microbes. Lignin will be decomposed. Finally, different microbe populations complete
the decomposition of different substrates and ultimately, all the organic matter. Thus,
microbes are substrate specific.
Some microbes may produce an enzyme in larger quantity than others. Their rate of
decomposition of a substrate will be more. Others cannot compete with these. This
ability to produce larger quantity of a particular enzyme is industrially exploited.
Activation and Inhibition
A same type of enzyme may be secreted by two different types of microorganisms. For
example, β-galactosidase enzyme is secreted by both bacteria and fungi. The bacterial
enzyme needs the cofactor cobalt for functioning. But, cobalt inhibits the enzyme
action in fungi.
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Enzyme Production Methodology


1) Isolation of Microorganisms
In culture media, different microorganisms are present together. One particular
microorganism may be needed for a particular purpose. So, the needed microbe has to
be isolated. The purposes for which microorganisms are isolated are
a. production of enzyme in high quantity and other metabolites less.
b. Short duration of fermentation process.
c. Low cost culture medium.
2) Development of Strains
The isolated microorganism should be made to produce more enzymes. For this, some
strains of the isolated microorganisms are improved to optimum level through
mutagens. The culture medium must also be suitably formulated.
3) Inoculum Preparation
The spores and mycelia of these strains are allowed to multiply on liquid broth.
Inoculum is prepared.
Medium Formulation and Preparation
Medium is formulated to increase enzyme production only. In the medium, the
following should be available from cheap sources: carbon, nitrogen, amino acids, growth
promoters, trace elements and a little salt. pH and temperature are also maintained at
optimum level.
Sterilization and inoculation of Medium, Maintenance of Culture and Fluid
Filtration
Fermenter should be large. The medium is sterilized continuously or batch-wise.
A sufficient amount of inoculum to start fermentation is inoculated. Submerged or
surface culture method is used.
The growth parameters like pH, temperature, etc. are maintained optimally. They are
different for different microbes. Oil may be added to prevent foaming during
fermentation. After 30 to 150 hrs. extra-cellular enzymes and other metabolites
accumulate in the medium.
After fermentation is over, the broth is kept at 50 C. to avoid contamination. The
bacterial broth is treated with calcium salts. Calcium phosphate gets precipitated.
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Bacterial cells and colloids are separated. The liquid is filtered and centrifuged. Cell
debris is removed.
Purification of Enzymes
It is a complicated process. The steps are,
• A concentrated solution is prepared by vacuum evaporation. Temperature is
kept low.
• Ultra-filtration can also be used.
• Other microbes are removed by polishing filtration.
• Preservatives or stabilizers like calcium salts, proteins, etc. are added.
• Enzymes are precipitated with acetone, alcohols or organic salts.
• The precipitate is dried.
• Packaged for marketing.
IMMOBILIZATION OF ENZYMES
Some enzymes produced by microorganisms are in small quantity. But, the production
cost is high. So, they have to be reused. For this, immobilization technique is
developed.
Definition of Immobilization
The physical attachment or localization of an enzyme in a defined region or matrix so
that it can be reused whenever needed is called immobilization of enzyme.
Advantages
• Continuous reuse.
• Cost effectiveness
• Minimum reaction time.
• Less contamination
• More stable.
• Improved process control.
• High enzyme-substrate ratio.
Enzyme Immobilization Methods
There are five methods. Some carriers or support materials are needed. They may be a
matrix system, a membrane or a solid surface.
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(I) Adsorption:
In this method, the enzyme is bonded to a carrier. Bonding may be on or inside of the
carrier. Some often-used carriers are aluminium oxide, clay (minerals), starch (organic),
modified sapharose and ion exchange resins. Low energy bonds like ionic interactions,
hydrogen bond, van der Waals forces, etc. are involved.
In external immobilization, the carrier should be very small (500 Å to 1 mm.). This gives
more surfaces for bonding. As enzymes do not enter into the carrier, the pore size is not
considered. Also, the internally adsorbed enzymes are protected from abrasion,
inhibitory bulk solutions and microbial attack. A more stable and active enzyme system
is achieved.
There are four ways for immobilization by adsorption.
(1) Static Process - The enzyme-containing solution is allowed to contact the
carrier.
(2) Dynamic Batch Process - The carrier is placed inside the enzyme-containing
solution and stirred well.
(3) The Reactor-loading Process - The reactor vessel is taken. The carrier is
placed inside it. The enzyme solution is then added. Both are mixed by
agitation.
(4) The Electro Deposition Process - The enzyme bath is taken. The electrodes
are kept inside. The carrier is placed near the electrode. The current is put on.
The enzyme migrates and adsorbs on the carrier.
(2) Covalent Bonding
Covalent bond is formed between the enzyme and the carrier surface. The enzyme may
have a specific site for covalent bonding. There are many methods of bonding. In the
presence of the enzyme’s substrate, the bonding is easy.
(3) Entrapment
There are many water soluble polymers. They have a matrix. The enzyme can be
physically trapped in it. Pore size can be adjusted to prevent enzyme escape. There are
different types.
(4) Cross-linking or Copolymerization
If the various molecules of an enzyme are covalently bonded with poly-functional
reagents, it is called cross-linking or copolymerization. It is cheap, simple and
105

commercially used. But, there is the chance of enzyme denature. This has also many
ways.
(5) Encapsulation
A drop of enzyme is enclosed inside a capsule of semi-permeable membrane. This is
simple, cheap and used in medicine. Large quantity of enzymes can be immobilized.
Immobilization of Cells
If the whole cell is immobilized, the enzymes are active and stable. The immobilization
methods for enzymes are applicable to cell immobilization too.
Enzyme Engineering
Enzymes are proteins. Genes code for enzymes. If the gene is changed, the enzyme
also changes. So, the enzymes can be engineered.
Applications of Enzymes
• Therapeutic - Enzymes can be used in disease curing. Treatments for leukaemia,
worm infections, bleeding, etc. are employed.
• Analytical - Enzymes are used in finding end points, kinetic analysis, etc.
Nowadays, it is used as a biosensor.
• Manipulative uses - They are used in genetic engineering.
• Industrial uses - They are used in dairy, detergent, starch, pharmaceutical and
wine industries.
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16. BIOSENSOR
Definition
A Biosensor is an analytical device consisting of an immobilized layer of biological
material (enzyme, antibody, etc.) in intimate contact with a transducer or sensor (a
physical component) which analyses the biological signals and converts them into
electrical signals.
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What can be a sensor?


Any physical component can be a sensor. Ex.: a single carbon electrode, an ion-sensitive
electrode, oxygen electrode, a photocell or a thermistor.
A biological material is immobilized on a permeable membrane. A sensor is kept near it.
The material crosses through the membrane. It reacts with the immobilized material.
The reaction produces a product. It passes through another membrane to the sensor.
The sensor transforms the product into electric signal. It is then amplified. Another
equipment processes this amplified signal into a display. This is read and recorded.
Glucose Biosensor
A platinum oxygen-electrode is taken. Polyacrylamide gel is placed around it. Both are
separated by a teflon membrane. Glucose oxidase is immobilized in the gel. It is
covered from the upper surface with cellulose acetate membrane. KCl solution is placed
around the electrode.
Glucose is placed over the membrane. Glucose and oxygen pass through the membrane
into the enzyme layer. Oxidation-reduction reaction takes place. Gluconic acid and
hydrogen peroxide are formed. Due to this, in the gel, oxygen concentration decreases.
Hydrogen peroxide effects a current change. This is the measurable signal. The
electrode records the rate of reactions. The rate of decrease of O2 concentration is
proportional to the glucose concentration. The Central Electro-Chemical Research
Institute, Karaikudi, has developed a glucose biosensor for as low as 0 to 15 millimoles.
Types of Biosensors
There are different types. They are based on the biological materials and sensor devices
used.
1. Electro-chemical Biosensor
2. Amperometric Biosensor
3. Thermistor-containing Biosensor
4. Bioaffinity Sensor
5. Whole cell / Microbial Biosensor
6. Opto-electronic Biosensor
Applications of Biosensors
Biosensors are small, fast and handled easily. So, they are used in (1) Medicine and
Health, (2) Pollution Control, (3) Industry, (4) Military, etc.
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BIOCHIPS or THE BIOLOGICAL COMPUTER


Formerly, the computers were costly, large and occupying large space. Later, silicon
microchips were developed. This changed the computer technology very much. Now,
microchips and biotechnology are combined to develop Biochips.
Protein Frame

Semi- conducting Molecule

Protein Support

Principle
Macromolecules like proteins shape themselves. They have a predetermined three-
dimensional structure. This is helpful in designing the biochips. The circuits can be
crammed around the three dimensional structure. The electrical signals can pass
through the semi-conducting organic molecule as in silicon microchips.
Design
A protein framework is formed. A semiconducting organic molecule is inserted into it.
This set-up is mounted on a protein support.
Advantages
1. Biochip is smaller than the microchip. The width of the electrical circuit is only
that of a protein molecule. This is smaller than the smallest silicon chip.
2. Electron tunneling is easier.
3. The protein molecule has less electrical resistance. Heat produced is less during
electrical signal production. So, many circuits can be placed together.
Applications
In artificial limbs, the nerve impulses are made to look more natural.
It can be used as a heart heat regulator. Using the costly pace-makers can be avoided.
It can sense light and sound and convert them into electrical signals. So, in future, the
blind can see and the deaf can hear.It is immune to the electromagnetic effects of
nuclear weapons. It can protect the silicon-based computers. So, it can be used in
military.
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Contents
1. CELL STRUCTURE ..................................................................................................... 1
2. BACTERIA ................................................................................................................ 3
3. EUKARYOTIC CELL ................................................................................................... 9
4. CELL DIVISION ....................................................................................................... 19
5. Meiosis ................................................................................................................. 25
6. Nucleic Acid – DNA - STRUCTURE .......................................................................... 29
7. CANCER ................................................................................................................ 36
8. MENDELIAN TRAITS IN MAN ................................................................................. 46
9. SYNDROMES ......................................................................................................... 60
10. BLOOD GROUPS, ANTIGEN-ANTIBODY REACTION .............................................. 69
11. Blood coagulation ............................................................................................. 73
12. REPRODUCTIVE CYCLE IN MAN .......................................................................... 81
13. CONSTRUCTION OF rDNA .................................................................................. 91
14. FERMENTATION ................................................................................................ 94
15. ENZYME TECHNOLOGY .................................................................................... 100
16. B I O S E N S O R .............................................................................................. 106

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