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The light microscope is a type of microscope which uses visible light and a system of lenses to magnify
images of small samples. There are two basic configurations of the conventional optical microscope, the
simple (one lens) and compound (many lenses). To focus at different focal depths the lens to sample
distance is adjusted and to get a wider or narrower field of view a different magnification objective lens
are used. A simple microscope is a microscope that uses only one lens for magnification, and is the
original design of light microscope. A compound microscope is a microscope which uses multiple lenses
to collect light from the sample and then a separate set of lenses to focus the light into the eye or
camera. Compound microscopes are heavier, larger and more expensive than simple microscopes due
to the increased number of lenses used in construction.
Construction
Ocular
The ocular, or eyepiece, is a cylinder containing two or more lenses; its function is to bring the image
into focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are
interchangeable and many different eyepieces can be inserted with different degrees of magnification.
Typical magnification values for eyepieces include 2×, 5× and 10×.
Objective
The objective is a cylinder containing one or more lenses that are typically made of glass; its function is
to collect light from the sample. At the lower end of the microscope tube one or more objective lenses
are screwed into a circular nose piece which may be rotated to select the required objective lens. Typical
magnification values of objective lenses are 4×, 5×, 10×, 20×, 40×, 50×, 60× and 100×.
Stage
The stage is a platform below the objective which supports the specimen being viewed. In the center of
the stage is a hole through which light passes to illuminate the specimen. The stage usually has arms to
hold slides.
Light source
Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes,
however, have their own controllable light source - normally a halogen lamp.
Frame
The whole of the optical assembly is traditionally attached to a rigid arm which in turn is attached to a
robust U shaped foot to provide the necessary rigidity. The arm angle may be adjustable to allow the
viewing angle to be adjusted.
The frame provides a mounting point for various microscope controls. Normally this will include controls
for focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled
wheel to control fine focus. Other features may be lamp controls.
Optical microscopy is used for medical diagnosis, the field being termed histopathology when
dealing with tissues, or in smear tests on free cells or tissue fragments.
In the bright field microscopy the image is a dark sample on a bright background. Bright field microscopy
typically has low contrast with most biological samples as few absorb light to a great extent. Stains are
often required to increase contrast which prevents use on live cells in many situations. Bright field
illumination is useful for samples which have an intrinsic colour, for example chloroplasts in plant cells.
Light Path
Transillumination light source, commonly a halogen lamp in the microscope stand
Condenser lens which focuses light from the light source onto the sample
Objective lens which collects light from the sample and magnifies the image
Advantages
Simplicity of setup with only basic equipment required.
Low apparent optical resolution due to the blur of out of focus material
The sample has to be stained before viewing. Therefore, live cells cannot be viewed
Dark field microscopy describes microscopy methods which exclude the unscattered light beam from
the image. As a result, the field around the specimen (i.e. where there is no specimen to scatter the
beam) is generally dark.
The light enters the sample. Most is directly transmitted, while some is scattered from the sample
The scattered light enters the objective lens, while the directly transmitted light simply misses the
lens and is not collected due to a direct illumination block
Only the scattered light goes on to produce the image, while the directly transmitted light is omitted
Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in
the light passing through a transparent specimen is converted into contrast changes in the image. A
phase contrast microscope does not require staining to view the slide. This type of microscope made it
possible to study the cell cycle.
Principle
As light travels through a medium its amplitude and phase to change in a way which depends on
properties of the medium. Changes in amplitude give rise to familiar absorption of light which gives rise
to colours which is wavelength dependent.
Fluorescence Microscope
The specimen is labeled with a fluorescent molecule called a fluorophore (such as green fluorescent
protein (GFP), fluorescein or DyLight 488). The specimen is illuminated with light of a specific
wavelength which is absorbed by the fluorophores.
Construction
Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor
lamp), the excitation filter, the dichroic mirror (or dichromatic beam splitter), and the emission filter.
The polarized light enters the first Nomarski-modified Wollaston prism and is separated into two
rays polarized at 90° to each other, the sampling and reference rays. Wollaston prisms are a type of
prism made of two layers of a crystalline substance, such as quartz, which, due to the variation of
refractive index depending on the polarisation of the light, splits the light according to its
polarisation. The Nomarski prism causes the two rays to come to a focal point outside the body of
the prism, and so allows greater flexibility when setting up the microscope, as the prism can be
actively focused.
The two rays are focused by the condenser for passage through the sample. These two rays are
focused so they will pass through two adjacent points in the sample.
The sample is effectively illuminated by two coherent light sources, one with 0° polarisation and the
other with 90° polarisation.
The rays travel will experience different optical path lengths where the areas differ in refractive
index or thickness. This causes a change in phase of one ray relative to the other due to the delay
experienced by the wave in the more optically dense material.
The rays travel through the objective lens and are focused for the second Nomarski-modified
Wollaston prism.
The second prism recombines the two rays into one polarized at 135°. The combination of the rays
leads to interference, brightening or darkening the image at that point according to the optical path
difference.
The main limitation of DIC is its requirement for a transparent sample of fairly similar refractive index to
its surroundings. DIC is unsuitable for thick samples, such as tissue slices, and highly pigmented cells. DIC
is also unsuitable for most non biological uses because of its dependence on polarisation, which many
physical samples would affect.
Atomic force microscopy is a very high-resolution type of scanning microscopy, with demonstrated
resolution on the order of fractions of a nanometer, more than 1000 times better than the optical
diffraction limit.
Basic Principles
The AFM consists of a cantilever with a sharp tip at its end that is used to scan the specimen surface.
The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of
nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the
sample lead to a deflection of the cantilever according to Hooke's law. Depending on the situation,
forces that are measured in AFM include mechanical contact force, van der Waals forces, capillary
forces, chemical bonding, electrostatic forces, magnetic forces, Casimir forces, solvation forces, etc.
Typically, the deflection is measured using a laser spot reflected from the top surface of the cantilever
into an array of photodiodes. Other methods that are used include optical interferometry, capacitive
sensing or piezoresistive AFM cantilevers. These cantilevers are fabricated with piezoresistive elements
that act as a strain gauge. Using a Wheatstone bridge, strain in the AFM cantilever due to deflection can
be measured, but this method is not as sensitive as laser deflection or interferometry.
Advantages
AFM has several advantages over the scanning electron microscope (SEM). Unlike the electron
microscope which provides a two-dimensional projection or a two-dimensional image of a sample, the
AFM provides a three-dimensional surface profile. Additionally, samples viewed by AFM do not require
any special treatments (such as metal/carbon coatings) that would irreversibly change or damage the
sample. While an electron microscope needs an expensive vacuum environment for proper operation,
most AFM modes can work perfectly well in ambient air or even a liquid environment. This makes it
possible to study biological macromolecules and even living organisms. In principle, AFM can provide
higher resolution than SEM. It has been shown to give true atomic resolution in ultra-high vacuum (UHV)
and, more recently, in liquid environments. High resolution AFM is comparable in resolution to scanning
tunneling microscopy and transmission electron microscopy.
Disadvantages
The scanning speed of an AFM is also a limitation. Traditionally, an AFM cannot scan images as fast as a
SEM, requiring several minutes for a typical scan, while a SEM is capable of scanning at near real-time,
although at relatively low quality.
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a
micrograph by using point illumination. It enables the reconstruction of three-dimensional structures
Basic Concept
A confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of
the detector to eliminate out-of-focus signal - the name "confocal" stems from this configuration.
Uses
CLSM is widely-used in numerous biological science disciplines, from cell biology and genetics to
microbiology and developmental biology.
Clinically, CLSM is used in the evaluation of various eye diseases, and is particularly useful for
imaging, qualitative analysis, and quantification of endothelial cells of the cornea. It is used for
localizing and identifying the presence of filamentary fungal elements in the corneal stroma in cases
of keratomycosis, enabling rapid diagnosis and thereby early institution of definitive therapy.
The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a
variety of signals at the surface of solid specimens. The SEM’s job is to use an electron beam to trace
over the object, creating an exact replica of the original object on a monitor. As the electron beam
traces over the object, it interacts with the surface of the object, dislodging secondary electrons from
the surface of the specimen in a unique pattern. A secondary electron detector attracts those scattered
electrons and, depending on the number of electrons that reach the detector, registers different levels
of brightness on a screen. The scanning electron microscope has many advantages over traditional
microscopes. The SEM has a large depth of field, which allows more of a specimen to be in focus at one
time, producing strikingly clear images. The SEM also has much higher resolution, so closely spaced
specimens can be magnified at much higher levels. Because the SEM uses electromagnets rather than
lenses, the researcher has much more control over the degree of magnification.
Lenses
SEMs use lenses to produce clear and detailed images but the lenses work differently and they are made
of magnets capable of bending the path of electrons. By doing so, the lenses focus and control the
electron beam, ensuring that the electrons end up precisely where they are needed.
Sample Chamber
The sample chamber of an SEM is where specimen is placed in a vacuum. Because the specimen must
be kept extremely still for the microscope to produce clear images, the sample chamber must be very
sturdy and insulated from vibration. In fact, SEMs are so sensitive to vibrations that they’re often
installed on the ground floor of a building. They also manipulate the specimen, placing it at different
angles and moving it so that researchers don’t have to constantly remount the object to take different
images.
Detectors
These devices detect the various ways that the electron beam interacts with the sample object. For
instance, Everhart-Thornley detectors register secondary electrons, which are electrons dislodged from
the outer surface of a specimen. These detectors are capable of producing the most detailed images of
an object’s surface. Other detectors, such as backscattered electron detectors and X-ray detectors, can
tell researchers about the composition of a substance.
Vacuum Chamber
Scanning coils create a magnetic field using fluctuating voltage, to manipulate the electron beam. The
scanning coils are able to move the beam precisely back and forth over a defined section of an object. If
a researcher wants to increase the magnification of an image, he or she simply sets the electron beam to
scan a smaller area of the sample.
Scanning Process
The electron beam is focused by condenser lenses to a spot. The beam passes through pairs of scanning
coils in the electron column which deflect the beam in the x and y axes so that it scans in a raster fashion
over a rectangular area of the sample surface.
The energy exchange between the electron beam and the sample results in the reflection of high-energy
electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the emission
of electromagnetic radiation, each of which can be detected by specialized detectors.
A source at the top of the microscope emits electrons that travel through vacuum in the column of the
microscope. Instead of glass lenses focusing is done by electromagnetic lenses and the electrons are
focussed into a very thin beam. The electron beam then travels through the specimen you want to
study. Depending on the density of the material present, some electrons are scattered and disappear
from the beam and unscattered electrons hit a fluorescent screen, which produces the image of the
specimen with its different parts displayed in varied darkness according to their density. The image can
be studied directly by the operator or photographed with a camera.
TEM Working
This stream is focused to a narrow beam by the use of condenser lenses.
The beam strikes the specimen and parts of it are transmitted.
This transmitted portion is focused by the objective lens into an image.
The image is passed down the column through the intermediate and projector lenses and is
enlarged all the way.
The image strikes the phosphorescent image screen and light is generated, allowing the user to see
the image. The darker areas of the image represent those areas of the sample that fewer electrons
were transmitted through, meaning that they are thicker or denser. The lighter areas of the image
represent those areas of the sample that more electrons were transmitted through, meaning that
they are thinner or less dense.
The spectrophotometer is an instrument which measures the amount of light of a specified wavelength
which passes through a medium. According to Beer's law, the amount of light absorbed by a medium is
proportional to the concentration of the absorbing material or solute present. Thus the concentration of
a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a
given wavelength. Wavelength (often abbreviated as lambda) is measured in nm. The
spectrophotometer allows selection of a wavelength pass through the solution. Usually, the wavelength
had chosen which corresponds to the absorption maximum of the solute. Absorbency is indicated with a
capital A.
Design
There are two major classes of devices: single beam and double beam. A double beam
spectrophotometer compares the light intensity between two light paths, one path containing a
reference sample and the other the test sample. A single beam spectrophotometer measures the
relative light intensity of the beam before and after a test sample is inserted. Although comparison
measurements from double beam instruments are easier and more stable, single beam instruments can
have a larger dynamic range and are optically simpler and more compact.
A fraction of the monochromatic light is transmitted through the sample and to the photodetector
How to use
Warm up: Plug in and turn on. Allow about 30 minutes for warm up
Blank adjust: Fill the B (blank) cuvette with the solvent used to dissolve specimen (often distilled
water). Polish to clean, insert into the cuvette chamber, aligning mark to front. Close chamber cover
Rotate blank adjust knob (front right knob) to adjust absorbance to read zero
Read specimen: Pour the sample into the S (specimen) cuvette, polish and insert into the chamber,
aligning mark to the front
Note that the scale for absorbance is the lower scale on the dial, and should be read from R to L
If you read additional specimens, you should confirm that the machine is still zeroed and blanked
out, as in steps 2, 4 and 5 for all readings
Clean up: Remove cuvette from machine, carefully wash and store spectrophotometer cuvettes
keeping them separate from regular test tubes.
Ultracentrifuge
The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of
generating acceleration as high as 1,000,000 g (9,800 km/s²). There are two kinds of ultracentrifuges,
the preparative and the analytical ultracentrifuge. Both classes of instruments find important uses in
molecular biology, biochemistry and polymer science.
A centrifuge of high or low speed which provides convection-free conditions and which is used for
quantitative measurement of sedimentation velocity or sedimentation equilibrium or for the separation
of solutes in liquid solution. See also Centrifugation.
The ultracentrifuge is used (1) to measure molecular weights of solutes and to provide data on
molecular weight distributions in polydisperse systems; (2) to determine the frictional coefficients, and
thereby the sizes and shapes, of solutes; and (3) to characterize and separate macromolecules on the
basis of their buoyant densities in density gradients. See also Molecular weight.
The ultracentrifuge is most widely used to study high polymers, particularly proteins, nucleic acids,
viruses, and other macromolecules of biological origin. However, it is also used to study solution
properties of small solutes. In applications to macromolecules, the analytical ultracentrifuge, which is
used for accurate determination of sedimentation velocity or equilibrium, is distinguished from the
preparative ultracentrifuge, which is used to separate solutes on the basis of their sedimentation
velocities or buoyant densities.
Apoptosis is of Greek origin, having the meaning "falling off or dropping off". Apoptosis is an energy
dependent process by which individual cells undergo programmed cell death in response to various
extrinsic and intrinsic factors without inducing inflammatory responses. Normal development of organ
requires not only cell division and cell differentiation but also elimination of cell by apoptosis. The term
programmed cell death was introduced in 1964, proposing that cell death during development is not of
accidental nature but follows a sequence of controlled steps leading to locally and temporally defined
self-destruction.
Induction Stage
In this stage there is initial signal for apoptosis by variety of stimuli like various stresses including
deprivation of serum, growth factors or cytokines, heat shock and various carcinogenic reagents.
Execution Stage
This stage involves the classic morphological and biochemical changes. Morphological changes includes
condensation and peripheralization of chromatin, vacuolization and loss of cytoplasm, fragmentation of
nucleus, corn paction of organelles, the disestablishment of communication with neighboring cells,
fusion of the endoplasmic reticulum with the outer cell membrane. The biochemical change involves the
activation of a specific series of cytoplasmic cellular proteases and endonucleases, caspase. The
activation of these self- catalytic caspase in the cytoplasm has been identified.
Degradation Stage
This occurs due to the various morphological and biochemical changes mentioned above. Finally there is
fragmentation of the cell DNA and other macromolecules into size of nucleosome units due to the
activation of endogenous nuclear endonucleases. Water is extracted from the cell resulting into marked
decrease in cell size and increase in density. The increase in density has been used to isolate apoptotic
from non-apoptotic cell. The shrunken apoptotic cells subsequently fragmented into sealed vesicles and
thus formation of numerous membranes bound apoptotic bodies containing DNA which are engulfed by
the surrounding cells.
During necrosis, the cellular contents are released uncontrolled into the cell's environment which results
in damage of surrounding cells and a strong inflammatory response in the corresponding tissue.
The DNA fragmentation which occurs in apoptosis can be visualized by agar gel electrophoresis. The
reduction in cell size and volume in apoptosis can be studied by microscopy of flow cytometry. A
number of dyes such as ethidium bromide or propidium iodide can enter in the apoptotic cell due to
altered permeability, and thus number of dead cells can be obtained in flow cytometry. The following
methods have been developed to quantify DNA fragmentation:
Sometimes through an active programme suppression of apoptosis is needed to maintain the cells in
quiescent state. The execution of the quiescence program appears to be essential for the long term
survival of peripheral lymphocytes and is dependent upon signals transduced through the Band T cell
antigen receptors. In addition, the transcription factor LKLF is required component of this programme in
T-cells.
Necrosis
Necrosis means the death of cells or tissues in the living body. Here cell dying by this process has no
control over its fate. In some cases death is rapid or sudden, but in others various degenerative changes
takes places, such as cloudy swelling and fatty degeneration, before the cell or tissues dies, and the
process of dying is gradual one. Here the cell death is due to leakage of the lysosomal enzymes into the
cytoplasm, swelling of the cell and eventual rupture of the plasma membrane. When a cell die in the
living body there follows short lapse of time during which comparatively little histological change takes
place and the cell presents an appearance identical with that shown by the healthy cell which has been
killed by fixation.
There are several causes, which initiates necrosis. These include followings:
Physical Agents
Various physical agents like electricity, extreme heat and cold and X-rays and prolonged pressure (by
ligature or tumors) may lead to necrosis of the cells.
Chemical Agents
Various chemicals such as carbolic acids, mineral acids and caustics act directly on cells resulting into
necrosis of cells.
Enzymes
Fat splitting enzymes from the pancreas which gives rise to fat necrosis.
Apoptosis
1. Apoptosis occurs at single cell level.
2. It is an active type of programmed cell death that is highly regulated and involves the activation of
cascade of molecular events leading to cell death.
3. It is a programmed cell death and is initiated as a response to external signals from other cells, or as a
result of changes in the intracellular macromolecules.
4. Here during cell death there is no leakage of the cell content, instead there is fragmentation of DNA,
the cytoplasm shows blabbing and increased granularity and there is fracturing of the cell into small
apoptotic bodies containing DNA.
Necrosis
1. Necrosis occurs in a group of cells or in tissues at a particular locus.
4. In necrosis cell death is due to leakage of the lysosomal enzymes into the cytoplasm, swelling of the
cell and eventual rupture of the plasma membrane.
During development many cells are produced in excess which eventually undergo programmed cell
death. For example:
During limb formation separate digits evolve by death of inter digital mesenchymal tissue.
Ablation of cells no longer needed such as the amphibian tadpole tail during metamorphosis.
Formation of reproductive organs e.g. process occurs in ovaries of the female when the number
of immature egg cells in the ovaries is gradually reduced from midway in pregnancy until birth.
Massive cell death occurs during early development of the nervous system. In the development
of the brain during which half of the neurons that is initially created will die in later stages when
the adult brain is formed.
Cells of an adult organism constantly undergo physiological cell death which must be balanced with
proliferation in order to maintain homeostasis in terms of constant cell number. Apoptosis involve in
the homeostasis of the immune system. Several millions of B and T cells are generated everyday and
the majority of those die during maturation (death by neglect, negative selection or by AICD of
peripheral immune cells).
Cells with severely damaged DNA that cannot be repaired appropriately usually are removed by
apoptosis
Inappropriate mitogenic signaling that is in conflict with the environmental or cellular status of the
cell usually results in cell cycle arrest or apoptosis
Elimination of infected cells Defects in apoptosis can result in cancer, autoimmune diseases and
spreading of viral infections, while neurodegenerative disorders, AIDS and ischemic diseases are
caused or enhanced by excessive apoptosis
It helps in removing the damaged, infected and potentially neoplastic cells and thus protects the
human beings and different livestock from various diseases including cancer.
Apoptosis in a cell can be triggered by verity of signals. This include a balance between the withdrawal
positive signals i.e. require for continue survival of the cell and the recipient of negative signals.
Apoptosis is a genetically controlled process and is controlled by certain genes. These genes either
inhibit or promote the apoptosis.
Promoters:
BAX
Fas
TP53
Inhibitors:
BCl-2
Pathway of Apoptosis
1. Intrinsic Pathway
This pathway also called as the mitochondrial pathway or granzyme pathway.
In this pathway the cytosol of T-lymphocytes secretes the granzyme and perforase.
The perforase make pores in the mitochondrial membrane and granzyme enter in mitochondria
through these pores.
Hare the granzyme make the release of cytochrome C.
The cytochrome C combines with the apoptosome which is formed by the Apaf-1 and
procaspase-1.
The apoptosome with cytochrome C act on inactive procaspase-13 and make it activated i.e.
caspase-13.
2. Extrinsic Pathway
In this pathway the inactive procaspase-8 is activated to caspase-8 by FADD.
The activated caspase-8 further act on inactive procaspase-3 and activated it to caspase-3 which
causes apoptosis.
When a cell received a signal that it is the time to die AIF is released from the mitochondria.
Apoptosis can be induced in response to various signals from inside and outside the cell. Signals
emanating from death receptors initially activate the Death Inducing Signaling Complex (DISC)
which mediates activation of the initiator caspase-8.
Activated caspase-8 initiates a caspase cascade by processing the effector caspases-3, -6, and –
7 which in turn cleave a number of protein substrates.
Cleavage of caspase substrate eventually leads to the characteristic morphological and
biochemical features of apoptosis.
In some cell systems, this direct caspase cascade is sufficient to elicit apoptosis on its own(type 1
signaling), whereas in other cases the signal coming from the DISC must be amplified by the
proteolytic activation of the BH3-only protein Bid by caspase-8 with subsequent induction of
apoptotic events at the mitochondria (type 2 signaling).
Mitochondrial apoptotic signaling includes the release of cytochrome c from the mitochondrial
inter membrane space to the cytosol where it contributes to the formation of the apoptosome
which consists of cytochrome c, Apaf-1 and dATP.
The apoptosome activates caspase-9 which is another initiator caspase and thus is able to
mediate the caspase cascade by activating caspase-3.
Another mitochondrial pro apoptotic factor is Smac which acts by inhibiting the IAPs from
blocking caspase activity.
IAPs are a family of proteins with anti-apoptotic activity by directly inhibiting caspase.
IAP expression can be up regulated in response to survival signals such as those coming from
growth factor receptors, e.g. by activation of the transcription factorNF-kB, therefore providing
a means to suppress apoptosis signaling.
Of central importance are the anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-XL
which counteract the action of BH3-only proteins such as Bid but also of pro apoptotic Bax and
Bak and thus can inhibit mitochondrial pro apoptotic events.
Apoptotic signals coming from the inside of the cell frequently have their origin within the
nucleus, being a consequence of DNA damage induced by irradiation, drugs or other sort of
stress.
DNA damage in most cases eventually results in the activation of the p53 transcription factor
which promotes expression of proapoptotic Bcl-2 members and suppresses anti apoptotic Bcl-2
and Bcl-XL. Other organelles besides mitochondria and the nucleus, such as the ER and
lysosomes also have been implicated in apoptotic signaling pathways, and it should be kept in
In a viable cell, the proapoptotic Bcl-2 family members Bax, Bak, and BH3-only proteins are
antagonized by anti-apoptotic members such asBcl-2.
In response to an apoptotic stimulus, BH3-only members are activated by transcriptional up
regulation (Bax, Noxa, and Puma), sub cellular relocalization (Bim, Bmf), dephosphorylation
(Bad), or proteolysis (Bid).
Activated BH3-only proteins prevent anti apoptotic Bcl-2 members from inhibiting proapoptotic
members.
In addition, they might directly induce a conformational change of Bax and Bak which
subsequently oligomerize and insert into the mitochondrial membrane where they form pores
either by themselves or by associating with the permeability transition pore complex.
In a normal growing viable cell, the p53protein is in a metastable state, i.e. p53 is susceptible to
targeted ubiquitination and subsequent proteasomal degradation.
Mdm2 directly interacts with p53 and thereby catalyzes ubiquitination ofp53.
Ubiquitination of p53 can be reversed by the action of the deubiquitinating enzyme HAUSP
which thereby can rescue p53 from degradation.
P53 is stabilized in response to genotoxic stress such as DNA damage which leads to the
phosphorylation of p53 at several specific serine and threonine residues.
Stabilized and activated p53 can translocate into the nucleus where it activates the transcription
of proapoptotic genes and suppresses the transcription of anti-apoptotic genes what under
certain conditions can result in the induction of apoptosis.
P53-mediated apoptosis signaling is dependent on the interplay of many regulatory factors,
including proto oncogenes as well as tumor-suppressors.
In all metazoan animals the life cycle includes two developmental periods
Post embryonic period or post natal development: It include growth, adulthood, reproduction, ageing
and death.
A slow deterioration in structure and functions of a body the cells, tissue and organ or a process that
make the organism more susceptible to disease after the attainment of reproductive maturity is called
as the ageing.
Gerontology
Geron=old man; logy=study; the branch of science deals with the study of ageing is called as
gerontology. It includes three aspects:
Biological Aspects: This category deals with the fundamental aspects of ageing. In this field we
studied the molecular, biochemical, biophysical and physiological changes that occur in an organism
after the attainment of reproductive maturity.
Clinical Aspects: This aspect concern with the old age diseases like cardiovascular,
cerebrovascular, arthritis, cancer and autoimmune diseases and methods for curing them.
Sociopsycological Aspects: This aspect concern with the Social and psychological problems of old
peoples.
Characteristics of Ageing
Adaptability is decreases.
Nonfunctioning of some vital organs like heart, kidney, liver and lungs etc. are happened.
Heart:
Efficiency of heart decreases with age so that heart pumps about 65% of blood per minute.
A Blood supply to various organs is decreases.
Oxygen consumption is decreases.
Lungs:
Vital capacity of lungs is decrease due to rigidity of chest wall.
Decrease lung elasticity and decline in capacity for gaseous exchange.
Inflammation happens in lung mucosa.
Kidney:
Number of nephrons is decreases.
Rate of gloumular filtration is decreases.
Urine output is decreases.
Blood:
Blood value is decreases as the rate of haemetapoisis is decreases.
Hemoglobin is decreases.
Cardiac output is decreases.
Blood pressure is decreases.
Skin:
It becomes dry as the water holding capacity of cells decreases and number of sweet glands is
decreases.
Digestive Organs:
Number of taste bud is decreases.
Secretion of digestive juices is decreases which cause problem like obesity, indigestion,
constipation and gases.
Teeth are lost due to degeneration of tooth forming tissue and supporting tissue.
Eyes:
Accommodation power of eyes is decreases.
Lens becomes opaque causing cataract.
Resolving power of eyes is decreases.
Thymus:
Thymus is decrease in size.
Less production of T-lymphocytes happens.
Immunity is decreases.
Autoimmune disease happens.
Ovary:
Follicle atresia is take place.
It is a degenerative process in which the follicle losses their structural integrity and oocytes are
lost from the ovary by other than the process of ovulation.
Follicle atresia results in extensive loss of germ cells during neonatal, prenatal, perinatal, aster
cycle, pregnancy, lactation and post reproductive life of mammals.
Hypotrophy i.e. decreases volume of body cells also decrease in size of nucleus.
Nuclear chromatin becomes condensed due to loss of water from the nucleoplasm and darkly
stained picnotic nucleus, phenomenon is called as picnosis.
Accumulation of pigments like lipofuscin (also called eye pigment formed by the unsaturated fatty
acids). It is the most distinguished and visible changes that occur in a non-dividing cell like neuron or
skeletal muscle cell. It also occurs in some dividing cells of liver, adrenal cortex and testis. Its
deposition is enhanced by administration of cortisol, by deficiency of vitamin E and by less supply of
oxygen under certain pathological conditions in early changes.
Semipermiability of cell membrane is decreases due to deposition of Ca++ ions in the peripheral part
of cytoplasm.
Rate of protein synthesis is decreases due to decrease in number of rough endoplasmic reticulum.
Increase in the amount of free radicles cause peroxidation of polyunsaturated fatty acids which
causes the destruction of the biological membrane due to which ageing take place.
This theory states that ageing results from damage to the genetic integrity of the body’s cells. The
somatic mutation is causes by the unfavorable environmental factors like cosmic rays and X-rays etc. if
the mutation occurs then it cause disturbed metabolism so formation of defective proteins results
declining metabolic rate causes ageing.
This theory was proposed by the M.S. Kanungo (1970). This theory states that ageing is an intrinsic
phenomenon controlled by some genetic time table called as ageing gene of genome. It induces at
specific period. E.g. annual plants age at specific period even in the most favorable conditions. This
theory explains two important characteristics of ageing:
This theory states that the organism which have higher metabolic rate will die earlier than the organism
which have low metabolic rate.
This theory states that the ageing is due to the accumulation of metabolic waste inside the body cells
which disturb the metabolism thus decline rate of metabolism results ageing.
It states that the brain is the biological clock which determines the ageing. Ageing is due to the changes
in certain parts of CNS which regulates the functioning of endocrine gland.
Abnormal secretion of neurohormone cause hormonal imbalance which causes malfunctioning of body
parts results in ageing.
It states that the ageing is due to the decrease in activity of immunological system of body and states
that the thymus is the biological clock of the man.
This theory was proposed by the F. Verzar. This theory states that the ageing is due to the changes in
collagen protein in the intestinal fluid surrounding the body cells.
This theory was proposed by the Orgelel. This theory states that the ageing is due to the error in
reading of genetic code results in the formation of the defective protein which form defective enzymes
which induce ageing. Such errors include incorporation of wrong nucleotides in mRNA during
transcription.
This very recent novel theory by Wang et al. suggests that ageing is the result of the accumulation of
"Misrepair". Important in this theory is to distinguish among "damage" which means a newly emerging
defect BEFORE any reparation has taken place, and "Misrepair" which describes the remaining defective
structure AFTER (incorrect) repair. The key points in this theory are:
There is no original damage left unrepaired in a living being. If damage was left unrepaired a life
threatening condition (such as bleeding, infection, or organ failure) would develop.
Misrepair, the repair with less accuracy, does not happen accidentally. It is a necessary measure of
the reparation system to achieve sufficiently quick reparation in situations of serious or repeated
damage, to maintain the integrity and basic function of a structure, which is important for the
survival of the living being.
Hence the appearance of Misrepair increases the chance for the survival of individual, by which the
individual can live at least up to the reproduction age, which is critically important for the survival of
species. Therefore the Misrepair mechanism was selected by nature due to its evolutionary
advantage.
However, since Misrepair as a defective structure is invisible for the reparation system, it
accumulates with time and causes gradually the disorganization of a structure (tissue, cell, or
molecule); this is the actual source of ageing.
Ageing hence is the side-effect for survival, but important for species survival. Thus Misrepair might
represent the mechanism by which organisms are not programmed to die but to survive (as long as
possible), and ageing is just the price to be paid.
A biosensor is an analytical device for the detection of an analyte that combines a biological component
with a physicochemical detector component. It consists of 3 parts:
The sensitive biological element (biological material, a biologically derived material or biomimic
e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.).
The sensitive elements can be created by biological engineering.
The transducer or the detector element (works in a physicochemical way; optical, piezoelectric,
electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte
with the biological element into another signal (i.e., transducers) that can be more easily
measured and quantified.
Associated electronics or signal processors that are primarily responsible for the display of the
results in a user-friendly way. This sometimes accounts for the most expensive part of the
sensor device. However it is possible to generate a user friendly display that includes transducer
and sensitive element.
A common example of a commercial biosensor is the blood glucose biosensor, which uses the enzyme
glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two electrons
to reduce the FAD to FADH2. This in turn is oxidized by the in a number of steps electrode by accepting
two electrons from the electrode. The resulting current is a measure of the concentration of glucose. In
this case, the electrode is the transducer and the enzyme is the biologically active component.
Many of today's biosensor applications are similar, in that they use organisms which respond to toxic
substances at a much lower concentration than humans can detect to warn of the presence of the toxin.
Such devices can be used in environmental monitoring, trace gas detection and in water treatment
facilities.
Procedure
The biological component interacts to analyte which produce some physical changes near transducer
surface. These physical changes may be:
Change in mass of biological components as a result of the reaction (Acoustic wave biosensor)
Nature of Interaction
The Nature of interaction between the analyte and biological material used in biosensor is of two types:
The analyte may be converted into a new chemical molecule by enzyme. Such biosensors are
called as the catalytic biosensor.
The analyte may be simply bind to the biological material present on the biosensor (e.g. to
antibodies, nucleic acid). Such biosensors are called as the affinity biosensor.
Properties of a Biosensor
The reaction used should be independent of manageable factors like the pH, temperature,
stirring etc.
The device should be tiny and bio compatible, in case it is to be used for analyses within the
body.
The device should be cheap, small, easy to use and capable of reuse.
Applications
There are many potential applications of biosensors of various types. The main requirements for a
biosensor approach to be valuable in terms of research and commercial applications are the
identification of a target molecule, availability of a suitable biological recognition element, and the
Environmental applications e.g. the detection of pesticides and river water contaminants
Detection of pathogens
Routine analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid as an
alternative to microbiological assay
Determination of drug residues in food such as antibiotics and growth promoters particularly in
meat and in honey
Cancer is the result of uncontrolled cell growth. In cancer, one of those cells stops paying attention to
the normal signals that tell cells to grow, stop growing or even to die.
Types Of cancer
Carcinomas: Cancer arising from epithelial cells or tissue, such as skin or epithelial lining of internal
organs or glands. It is about 85% of all the cancers. For example cancer of colon, breast, lung and
prostate.
Sarcomas: It is derived from the tissue of mesodermal origin such as bone, fat and cartilage. It is
very rare in humans and about 1% of all cancers.
Leukemia’s and lymphomas: It is the tumor of all the haematopoitic cells of bone marrow.
Leukemia’s proliferate as single cell and lymphomas tend to grow as tumor mass.
Tumor
Uncontrolled growth of cells results in tumors. There are two types of tumors:-
Begin tumor: It is usually grows of expansion and remains encapsulated in a layer of connective
tissue. It is not life threatening and are not considered as cancer. For example moles and warts.
Malignant tumor: It is characterized by uncontrolled proliferation and infinitive life span of the
proliferating cells. It becomes progressively invasive. It is life threatening and refer to as true cancer.
For example lung cancer and leukemia.
A wide range of changes occur during the transformation of a normal cell to a cell capable of forming a
cancerous growth. All cancer cells acquire the ability to grow and divide in the absence of appropriate
signals and/or in the presence of inhibitory signals. There are also detectable changes in the physical
properties of the cells. These changes include the following:
Cytoskeleton changes: The distribution and activity of the microfilaments and microtubules may
change. These alterations change the ways in which the cell interacts with neighboring cells and
alter the appearance of the cells. Changes in the cytoskeleton also affect cell adhesion and
movement (motility).
Nuclear changes: The shape and organization of the nuclei of cancer cells may be markedly
different from that of the nuclei of normal cells of the same origin. This change in appearance may
be useful in the diagnosis and staging of tumors.
Enzyme production: Cancer cells often secrete enzymes that enable them to invade neighboring
tissues. These enzymes digest away the barriers to migration and spread of the tumor cells.
Angiogenesis
Most tumors induce the formation of new blood vessels that invade the tumor and nourish it, a
process called angiogenesis.
Angiogenesis begins when a tumor becomes large enough where it needs increase the supply of
nutrients and oxygen it receives. Low oxygen levels (hypoxia) trigger the tumor and its surrounding
environment to release signals that result in the growth of blood vessels towards and into the
tumor. These new vessels supply oxygen and nutrients that allow the tumor to continue growing.
Because the development of blood vessels is so important in tumor growth therefore it is a target of
some cancer treatments.
Metastasis
The spread of cancer from its original location to other parts of the body is called metastasis.
Normally, loss of adhesion molecules results in their death; this is a protective mechanism that helps
prevent metastasis. Metastatic cells have developed mutations that allow them to disable this
mechanism and survive in the absence of adhesion.
In the Blood
Once tumor cells have successfully entered the blood they face an entirely new challenge: surviving
transit in the blood. Most cells, including tumor cells, are not designed to survive the stresses of the
blood system. One of the primary ways in which tumor cells survive this transit is by co-opting
platelets to act as shields. Platelets surround the cancer cells and protect them from both the force
of blood flow and attacks from immune cells.
In cancer cells, changes to key genes cause the cells to act abnormally. The changes are often the result
of changes to the DNA (mutations) in the cells.
The initial experimental studies of carcinogenesis were conducted in animals. Chemicals able to react
with DNA and non-reactive compounds were both tested for their ability to cause cancer. The model
used was mouse skin carcinogenesis. In this system researchers painted test chemicals on the skin and
observed the growth of tumors.
Researchers found that application of a DNA reactive substance only resulted in tumor formation when
the animals were further treated with another non-reactive substance.
A compound that reacts with DNA and somehow changes the genetic makeup of the cell is called a
mutagen.
The mutagens that predispose cells to develop tumors are called initiators and the non-reactive
compounds that stimulate tumor development are called promoters. Approximately 70% of known
mutagens are also carcinogens--cancer-causing compounds. A compound that acts as both an initiator
and a promoter is referred to as a 'complete carcinogen' because tumor development can occur without
the application of another compound.
The development of cancer takes place in a multi-step process. As the cells become more abnormal,
they gain new capabilities, such as the ability to release growth factors and digestive enzymes. The cells
continue to divide, impacting nearby normal cells, often reducing the function of the affected organ.
Hyperplasia: The altered cell divides in an uncontrolled manner leading to an excess of cells in that
region of the tissue. The cells have a normal appearance but there are too many of them!
Carcinoma in situ: Additional changes make the cells and tissues appear even more abnormal. The
cells are now spread over a larger area and the region of the tissue involved contains primarily
altered cells. The cells often 'regress' or become more primitive in their capabilities. An example
would be a liver cell that no longer makes liver-specific proteins. Cells of this type are said to be de-
differentiated or anaplastic. A key facet of in situ growths is that the cells are contained within the
initial location and have not yet crossed the basal lamina to invade other tissues. Cancers of this
type are often totally curable by surgery since the abnormal cells are all in one location.
Tumors of this type have not yet invaded neighboring tissue. Based on information about patients
with similar growths and microscopic examination, these growths are often considered to have the
potential to become invasive and are treated as malignant growths.
Cancer (Malignant tumors): These tumors have the ability to invade surrounding tissues and/or
spread (metastasize) to areas outside the local tissue. These metastatic tumors are the most
dangerous and account for a large percentage of cancer deaths.
The next few pages will go into some detail on the changes and capabilities that allow cancer cells to
form large tumors and to metastasize to other parts of the body.
Some tumors do not progress to the point where they invade distant tissues. Such tumors are said
to be benign. Because they do not spread beyond their initial location, they are not considered to be
cancerous. Benign tumors are less often lethal than malignant tumors, but they can still cause
serious health problems. Large benign tumors can put pressure on organs and cause other
problems. In the case of brain tumors, the limited space within the skull means that a large growth
in the brain cavity can be fatal.
Cancer-critical genes are those genes whose mutation contributes to the causation of cancer. The cell
division process is dependent on a tightly controlled sequence of events. These events are dependent
on the proper levels of transcription and translation of certain genes. When this process does not occur
properly, unregulated cell growth may be the end result. Of the 30,000 or so genes that are currently
thought to exist in the human genome, there is a small subset that seems to be particularly important in
the prevention, development, and progression of cancer. These genes have been found to be either
malfunctioning or non-functioning in many different kinds of cancer.
The genes that have been identified to date have been categorized into two broad categories,
depending on their normal functions in the cell.
Genes whose protein products can directly or indirectly prevent cell division or lead to cell death.
The normal versions of genes in the first group are called proto-oncogenes. The mutated or otherwise
damaged versions of these genes are called oncogenes.
Tumor suppressors function in many key cellular processes including the regulation of transcription,
DNA repair and cell: cell communication. The loss of function of these genes leads to abnormal cellular
behavior.
Oncogene
Proto-oncogene genes are normally promoting cell division. These genes can be “on” or “off” and when
they are “on” they promote cell division. To stop mitosis these gene or their gene products must be
inactivated. If these gene become permanently switched on then the uncontrolled cell division occur
which leads to the tumor formation. Mutant form of proto-oncogene is called as the oncogene. The
activation of proto-oncogene to oncogene is an important step in causing of cancer.
Point mutation (i.e., change in a single base pair) in a proto-oncogene that results in a
constitutively active protein product
Chromosomal translocation that fuses two genes together to produce a hybrid gene
encoding a chimeric protein whose activity, unlike that of the parent proteins, often is
constitutive
Chromosomal translocation that brings a growth regulatory gene under the control of a
different promoter that causes inappropriate expression of the gene
DNA segment including a proto-oncogene so that numerous copies exist leading to overproduction
of the encoded protein.
Intracellular proteins that regulate or inhibit progression through a specific stage of the cell
cycle (e.g., p16 and Rb)
When expressed these genes halt the passage through the cell cycle and prevent mitosis.
For cell division to occur these genes or their gene products must be inactive or absent. If the tumor
suppressor gene becomes permanently inactive or deleted through mutation the control over cell
division is lost and the mutant cells begin to proliferate in an uncontrolled fashion. A cell with it’s fully
complement of tumor suppressor gene is thought to be protected against effect of oncogenes. In most
cases loss of tumor suppressor function must be accompanied by the conversion of proto-oncogene to
oncogene before the cell become fully malignant.
pRB Gene
The retinoblastoma (RB) gene located on chromosome 13 encodes a protein denoted as pRB. This
protein is found in the nuclei of all cells and tissue types and also is found in both resting (Go) cells and
actively dividing cells. pRB is present at all stages of the cell cycle and is part of the regulatory pathway.
Retinoblastoma arises when both copies of the RB gene are inactivated. In the inherited form of the
disease one parental chromosome carries an alteration in this region usually a deletion. Somatic events
in retinal cell that cause loss of the other copy of the RB gene cause a tumor. Mutated RB gene fails to
encode for protein pRB which play a primary role in regulating passage of cells from G1 to s phase
during which DNA synthesis occurs.
p53 Gene
Another tumor suppressor gene p53 encode a nuclear transcription factor. Activation of p53 protein can
lead to several responses including DNA repair, cell cycle arrest and apoptosis. Elimination of p53
function is an important step in the progression of many cancer cells towards the fully malignant state.
p53 refer to as “guardian of genome”. Cell division does not normally require the involvement of p53. If
the DNA of a cell becomes damage as a result of exposure to mutagens the level of p53 rises and act
either to arrest the progression of the cell through G1 or to direct the cell towards apoptosis. If both
copies of p53 gene are inactivated the cell loses the ability to arrest the cell cycle or commit the cell an
abnormal chromosome complement which may lead to the formation of a malignant growth.
The assembly of distinct tissues and their organization into organs are determined by molecular
interactions at the cellular level and would not be possible without the temporally and spatially
regulated expression of a wide array of adhesive molecules. Cells in tissues can adhere directly to one
another (cell–cell adhesion) through specialized integral membrane proteins called cell-adhesion
molecules (CAMs) that often cluster into specialized cell junctions.
Cells in animal tissues also adhere indirectly (cell–matrix adhesion) through the binding of adhesion
receptors in the plasma membrane to components of the surrounding extra cellular matrix (ECM), a
complex interdigitating meshwork of proteins and polysaccharides secreted by cells into the spaces
between them.
Cell-Adhesion Molecules
A large number of CAMs fall into four major families: the cadherins, immunoglobulin (Ig) superfamily,
integrins, and selectins.
Many CAMs are mosaics of which can be found in more than one kind of CAM. They are called “repeats”
when they exist multiple times in the same molecule of many CAMs mediate, through their extracellular
domains, adhesive interactions between cells of the same type (homotypic adhesion) or between cells
of different types (heterotypic adhesion).
A CAM on one cell can directly bind to the same kind of CAM on an adjacent cell (homophilic binding) or
to a different class of CAM (heterophilic binding). CAMs can be broadly distributed along the regions of
plasma membranes that contact other cells or clustered in discrete patches or spots called cell junctions.
The cytosol-facing domains of CAMs recruit sets of multifunctional adapter proteins. These adapters act
as linkers that directly or indirectly connect CAMs to elements of the cytoskeleton
The formation of many cell–cell adhesions entails two types of molecular interactions. First, CAMs on
one cell associate laterally through their extracellular domains or cytosolic domains or both into
homodimers or higher-order oligomers in the plane of the cell’s plasma membrane; these interactions
are called intracellular, lateral, or cis interactions. Second, CAM oligomers on one cell bind to the same
or different CAMs on an adjacent cell; these interactions are called intercellular or trans interactions.
The cadherins are the major CAMs responsible for Ca++ dependent cell-cell adhesion in vertebrate
tissues. The first three cadherins that were discovered were named according to the main tissues in
which they were found: E cadherin is present on many types of epithelial cells; N-cadherin on nerve,
muscle, and lens cells; and P-cadherin on cells in the placenta and epidermis. These and other classical
cadherins are related in sequence throughout their extracellular and intracellular domains. There are
also a large number of non-classical cadherins, with more than 50 expressed in the brain alone. The non-
classical cadherins include proteins with known adhesive function, such as the desmosomal cadherins.
Together, the classical and non-classical cadherin proteins constitute the cadherin superfamily.
Each classical cadherin contains a single transmembrane domain, a relatively short C-terminal cytosolic
domain, and five extracellular “cadherin” domains. The extracellular domains are necessary for Ca++
binding and cadherin-mediated cell–cell adhesion. The Ca++ binding sites, located between the cadherin
repeats, serve to rigidify the cadherin oligomers. The cadherin oligomers subsequently form intercellular
complexes to generate cell–cell adhesion and then additional lateral contacts, resulting in a “zippering
up” of cadherins into clusters. In this way, multiple low-affinity interactions sum to produce a very tight
intercellular adhesion.
The C-terminal cytosolic domain of classical cadherins is linked to the actin cytoskeleton by a number of
cytosolic adapter proteins
Three possibilities are (1) in homophilic binding, molecules on one cell bind to other molecules of the
same kind on adjacent cells; (2) in heterophilic binding, the molecules on one cell bind to molecules of a
different kind on adjacent cells; (3) in linker-dependent binding, cell surface receptors on adjacent cells
are linked to one another by secreted multivalent linker molecules.
As their name implies, neural CAMs are of particular importance in neural tissues. One type, the NCAMs,
primarily mediate homophilic interactions.
An NCAM comprises an extracellular region with five Ig repeats and two fibronectin type III repeats, a
single membrane- spanning segment, and a cytosolic segment that interacts with the cytoskeleton. In
contrast, the extracellular region of L1-CAM has six Ig repeats and four fibronectin type III repeats. As
with cadherins, cis (intracellular) interactions and trans (intercellular) interactions probably play key
roles in IgCAM mediated adhesion.
Early electron micrographs of virtually all animal cells that were in contact revealed sites of cell–cell
contact with a characteristic intercellular gap. The most important feature of these junctions is not the
gap itself but a well-defined set of cylindrical particles that cross the gap and compose pores connecting
the cytoplasms of adjacent cells—hence their alternate name of intercytoplasmic junctions. In many
tissues (e.g., the liver), large numbers of individual cylindrical particles cluster together in patches. Ions,
many low-molecular-weight precursors of cellular macromolecules, products of intermediary
metabolism, and small intracellular signaling molecules can pass from cell to cell through gap junctions.
Structure
Vertebrate gap junctions are composed of connexins A completely different family of proteins, the
innexins, forms the gap junctions in invertebrates. Each vertebrate hexagonal particle consists of 12
connexin molecules: 6 of the molecules are arranged in a connexon hemichannel—a hexagonal cylinder
in one plasma membrane—and joined to a connexon hemichannel in the adjacent cell membrane,
forming the continuous aqueous channel between the cells. Each connexin molecule spans the plasma
membrane four times; one conserved transmembrane alpha helix from each subunit apparently lines
the aqueous channel. There are probably more than 20 different connexin genes in vertebrates, and
different sets of connexins are expressed in different cell types.
This diversity in channel composition leads to differences in the permeability of channels to various
molecules. The permeability of gap junctions can be altered by changes in the intracellular pH and Ca2+
concentration, as well as by the phosphorylation of connexin, providing numerous mechanisms for
regulating transport through them.
Gap junctions are also present in many non-neuronal tissues where they help to integrate the
electrical and metabolic activities of many cells. In the heart, for instance, gap junctions rapidly pass
ionic signals among muscle cells and thus contribute to the electrically stimulated coordinate
contraction of cardiac muscle cells during a beat.
Some extracellular hormonal signals induce the production or release of small intracellular signaling
molecules called second messengers (e.g., cyclic AMP and Ca2+) that regulate cellular metabolism.
Because second messengers can be transferred between cells through gap junctions, hormonal
stimulation of one cell can trigger a coordinated response by that same cell and many of its
neighbors.
Gap junctions play critical roles in the development of egg cells in the ovary by mediating the
movement of both metabolites and signaling molecules between an oocyte and its surrounding
granulosa cells as well as between neighboring granulosa cells.
Transmembrane receptors span the cell membrane, with part of the receptor outside and part inside
the cell. The chemical signal binds to the outer portion of the receptor, changing its shape and
conveying another signal inside the cell. Some chemical messengers, such as testosterone, can pass
through the cell membrane, and bind directly to receptors in the cytoplasm or nucleus.
Sometimes there is a cascade of signals within the cell. With each step of the cascade, the signal can be
amplified, so a small signal can result in a large response. Eventually, the signal creates a change in the
cell, either in the expression of the DNA in the nucleus or in the activity of enzymes in the cytoplasm.
These processes can take milliseconds (for ion flux), minutes (for protein- and lipid-mediated kinase
cascades), hours, or days (for gene expression).
All cells are equipped with elaborate systems for receiving signals from their environment. The
mechanisms involved in cell signaling are particularly crucial for understanding multi-cellular
organisms, and are an important in many diseases. Signaling systems are providing new targets for
treating disease.
Cells receive signals from their environment (light, odor, sound, nutrients) and other cells (divide,
differentiate, migrate, and transmit signals).
Signaling involves a receptor, the transduction of information from the cell surface to inside the cell,
and subcellular systems that make a response.
Receptors are proteins with specific binding sites for signaling molecules called ligands. Membrane
soluble ligands include steroid hormones (estrogen) which bind to cytoplasmic receptors. Most
other signaling molecules (peptides, proteins, gases, ions, nucleotides) bind to integral membrane
proteins.
Types of receptors include ion channels that can open or close (gated) allowing ions to enter or
leave cells, enzyme linked that activate endogenous or exogenous protein kinases (phosphate
transferring enzymes), and serpentine or G-protein-linked.
Ligand binding to receptor results in the production of second messengers (cAMP, cGMP, IP3, Ca++,
DAG, and NO). Activation frequently involves protein kinase cascades, a series of amplification steps
resulting in a rapid response.
Cells can have similar receptors, but make very different responses to the same ligand. The response
made depends on the availability of targets. One cell may have target proteins that lead to altered
transcription, while another has target proteins that lead to cell differentiation or division.
Signaling molecules
Most signal transduction involves the binding of extracellular signaling molecules (and ligands) to cell-
surface receptors. Such receptors typically face outward from the plasma membrane while triggering
events inside the cell. These molecules have been functionally classified as:
Kinds of Signals
Autocrine signals
Paracrine signals
Exocrine signaling
Exocrine signaling occurs when cells secrete signaling molecules into the blood. For example, the ovaries
in females and the testes in males are stimulated by hormones produced by the brain.
Synaptic signaling
Synaptic signaling is similar to paracrine signaling but there is a special structure called the synapse
between the cell originating and the cell receiving the signal. Synaptic signaling only occurs between
cells with the synapse; for example between a neuron and the muscle that is controlled by neural
activity.
G-protein-coupled receptors
G-protein-coupled receptors (GPCRs) are a family of integral membrane proteins that possess seven
membrane-spanning domains, and are linked to a guanine nucleotide-binding protein. Many receptors
make up this family, including adrenergic receptors, neurotransmitter receptors, olfactory receptors,
opioid receptors, chemokine receptors, and rhodopsin.
Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor. An inactive G
protein exists as a heterotrimer, a molecule composed of three different protein subunits: Gα, Gβ, and
Gγ. Once the GPCR recognizes a ligand, the shape (conformation) of the receptor changes to
mechanically activate the G protein, and causes one subunit (Gα) to bind a molecule of GTP (causing
activation) and dissociate from the other two G-protein subunits (Gβ and Gγ). The dissociation exposes
sites on the G-protein subunits that interact with other molecules. The activated G protein subunits
detach from the receptor and initiate signaling from many downstream effector proteins.
General structure of G protein coupled receptors consist of seven transmembrane -helical regions (H1–
H7), fourextracellular segments (E1–E4), and four cytosolic segments (C1–C4). The carboxyl-terminal
segment (C4), the C3 loops, and, in some receptors, also the C2 loop are involved in interactions with a
coupled trimeric G protein.
Cytokine receptors
The cytosolic domain of cytokine receptors associates with a separate JAK kinase. Ligand binding causes
a conformational change that promotes formation of a functional dimeric receptor, bringing together
two intrinsic or associated kinases, which then phosphorylate each other on a tyrosine residue in the
activation lip Phosphorylation causes the lip to move out of the kinase catalytic site, thus allowing ATP
or a protein substrate to bind. The activated kinase then phosphorylates other tyrosine residues in the
receptor’s cytosolic domain. The resulting phosphotyrosines function as docking sites for various signal-
transduction proteins.
The JAK-STAT signaling pathway transmits information from chemical signals outside the cell, through
the cell membrane, and into gene promoters on the DNA in the cell nucleus, which causes DNA
transcription and activity in the cell. The JAK-STAT system is a major signaling alternative to the second
messenger system. The JAK-STAT system consists of three main components: a receptor, JAK and STAT.
JAK is short for Janus Kinase, and STAT is short for Signal Transducer and Activator of Transcription. The
receptor is activated by a signal from interferon, interleukin, growth factors, or other chemical
messengers. This activates the kinase function of JAK, which autophosphorylates itself. The STAT protein
then binds to the phosphorylated receptor. STAT is phosphorylated and translocates into the cell
nucleus, where it binds to DNA and promotes transcription of genes responsive to STAT.
In mammals, there are seven STAT genes, and each one binds to a different DNA sequence. STAT binds
to a DNA sequence called a promoter, which controls the expression of other DNA sequences. This
affects basic cell functions, like cell growth, differentiation and death. Disrupted or deregulated JAK-
STAT functionality (which is usually by inherited or acquired genetic defects) can result in immune
deficiency syndromes and cancers.
Mechanism:
The binding of the ligand to the receptor triggers activation of JAKs. With increased kinase activity, they
phosphorylate tyrosine residues on the receptor and create sites for interaction with proteins that
contain phosphotyrosine-binding SH2 domains. STATs possessing SH2 domains capable of binding these
phosphotyrosine residues are recruited to the receptors, and are themselves tyrosine-phosphorylated
The pathway is negatively regulated on multiple levels. Protein tyrosine phosphatases remove
phosphates from cytokine receptors and activated STATs. More recently identified Suppressors of
Cytokine Signaling (SOCS) inhibit STAT phosphorylation by binding and inhibiting JAKs or competing with
STATs for phosphotyrosine binding sites on cytokine receptors. STATs are also negatively regulated by
Protein Inhibitors of Activated STATs (PIAS), which act in the nucleus through several mechanisms.
The MAPK/ERK pathway is a chain of proteins in the cell that communicates a signal from a receptor on
the surface of the cell to the DNA in the nucleus of the cell. The signal starts when a growth factor binds
to the receptor on the cell surface and ends when the DNA in the nucleus expresses a protein and
produces some change in the cell, such as cell division. The pathway includes many proteins, including
MAPK which is originally called ERK (Extracellular Signal Regulated Kinases)), which communicate by
adding phosphate groups to a neighboring protein, which acts as an "on" or "off" switch. When one of
the proteins in the pathway is mutated, it can be stuck in the "on" or "off" position, which is a necessary
step in the development of many cancers.
The binding of ligands (“first messengers”) to many cell surface receptors leads to a short-lived increase
(or decrease) in the concentration of certain low-molecular-weight intracellular signaling molecules
termed second messengers. Their concentration can be increased or decreased due to binding of ligands
to the cell surface receptors. The increased concentration of one or more second messenger triggers a
rapid alteration in the activity of one or more enzymes or nonenzymatic proteins.
3’, 5’- cyclic GMP (cGMP): Activates PKG (Protein Kinase G) and open ion channels in rod cells
Ca++ Ions: Involve in muscle contraction, Induced exocytosis of secretory vesicles in endocrine cells of
neurotransmitter vesicle of nerve cells
Phosphoinositides: Binds cytosolic proteins to plasma membrane and regulate signals that binds to
the Receptor Tyrosine Kinase (RTK)
Formation of cAMP:
In the adipose cells hormone-induced activation of adenylyl cyclase result in the formation of cAMP.
Role of cAMP
The effect of cAMP is mediated by the PKA. The increased concentration of cAMP activates the PKA and
vice versa. Inactive PKA is a tetramer consisting of two regulatory (R) subunits and two catalytic (C)
subunits. Each R subunit has two distinct cAMP-binding sites; binding of cAMP to both sites in an R
subunit leads to release of the associated C subunit, unmasking its catalytic site and activating its kinase
activity. Binding of cAMP by an R subunit occurs in a cooperative fashion; that is, binding of the first
cAMP molecule lowers the Kd for binding of the second. Thus small changes in the level of cytosolic
factor CREB
For example consider the operational model for rhodopsin-induced closing of cation channels in rod
cells:
In dark-adapted rod cells, a high level of cGMP keeps nucleotide-gated nonselective cation channels
open. Light absorption generates activated opsin, O*
This binds inactive GDP-bound Gt protein and mediates replacement of GDP with GTP
The free Gt ·GTP generated then activates cGMP phosphodiesterase (PDE) by binding to its
inhibitory subunits and dissociating them from the catalytic alpha and beta subunits
Relieved of their inhibition, the alpha and beta subunits convert cGMP to GMP
The resulting decrease in cytosolic cGMP leads to dissociation of cGMP from the nucleotide-gated
channels in the plasma membrane and closing of the channels
Let consider the example of IP3/DAG pathway and the elevation of cytosolic Ca++:
This pathway can be triggered by ligand binding to certain G protein–coupled receptors and several
other receptor types, leading to activation of phospholipase C
Cleavage of PIP2 by phospholipase C yields IP3 and DAG
After diffusing through the cytosol, IP3 interacts with and opens Ca++ channels in the membrane of
the endoplasmic reticulum causing release of stored Ca++ ions into the cytosol
One of various cellular responses induced by a rise in cytosolic Ca++ is recruitment of protein kinase C
(PKC) to the plasma membrane where it is activated by DAG
The activated kinase can phosphorylate various cellular enzymes and receptors, thereby altering
their activity
As endoplasmic reticulum Ca++ stores are depleted, the IP3-gated Ca++ channels bind to and open
store-operated TRP Ca++ channels in the plasma membrane, allowing influx of extracellular Ca++
The regulation of contractility of arterial smooth muscle is done by the nitric oxide (NO) and cGMP as
follow:
Nitric oxide is synthesized in endothelial cells in response to acetylcholine and the subsequent
elevation in cytosolic Ca++
NO diffuses locally through tissues and activates an intracellular NO receptor with guanylyl cyclase
activity in nearby smooth muscle cells
The resulting rise in cGMP leads to activation of protein kinase G (PKG), relaxation of the muscle,
and thus vasodilation
The cell-surface receptor for atrial natriuretic factor (ANF) also has intrinsic guanylyl cyclase activity
Stimulation of this receptor on smooth muscle cells also leads to increased cGMP and subsequent
muscle relaxation
This is the irreversible pathways in which a component is proteolytically cleaved. An example of such
pathway is the NF-kB pathway, which enables cells to respond immediately and vigorously to a number
of stress-inducing conditions. This mechanism involves phosphorylation and subsequent ubiquitin-
mediated degradation of an inhibitor protein and exemplified by the NF-kB transcription factor.
Mechanism:
In resting cells, the dimeric transcription factor NF-kB, composed of p50 and p65, is sequestered in
the cytosol, bound to the inhibitor I-kB.
Stimulation by TNF alpha or IL-1 induces activation of TAK1 kinase leading to activation of the
trimeric I-kB kinase.
Ionizing radiation and other stresses can directly activate I-kB kinase by an unknown mechanism.
Following phosphorylation of I-kB by I-kB kinase and binding of E3 ubiquitin ligase,
polyubiquitination of I-kB targets it for degradation by proteasomes.
The removal of I-kB unmasks the nuclear-localization signals (NLS) in both subunits of NF-kB,
allowing their translocation to the nucleus.
Here NF-kB activates transcription of numerous target genes, including the gene encoding the alpha
subunit of I-kB, which acts to terminate signaling.
The extracellular subunit of Notch on the responding cell is noncovalently associated with its
transmembrane-cytosolic subunit
Binding of Notch to its ligand Delta on an adjacent signaling cell first triggers cleavage of Notch by
the membrane-bound metalloprotease TACE (tumor necrosis factor alpha converting enzyme),
releasing the extracellular segment
Presenilin 1, an integral membrane protein, then catalyzes an intramembrane cleavage that releases
the cytosolic segment of Notch
Following translocation to the nucleus, this Notch segment interacts with several transcription
factors that act to affect expression of genes that in turn influence the determination of cell fate
during development
The cell cycle entails an ordered series of macromolecular events that lead to cell division and the
production of two daughter cells each containing chromosomes identical to those of the parental cell.
Duplication of the parental chromosomes occurs during the S phase of the cycle, and one of the
resulting daughter chromosomes is distributed to each daughter cell during mitosis. The control of the
events of the cell cycle ensures that the replication of chromosomes and their segregation to daughter
cells occur in the proper order.
Regulation of the cell cycle is critical for the normal development of multicellular organisms, and loss of
control ultimately leads to cancer. The master controllers of these events are a small number of
heterodimeric protein kinases that contain a regulatory subunit (cyclin) and catalytic subunit (cyclin
dependent kinase). These kinases regulate the activities of multiple proteins involved in DNA replication
and mitosis by phosphorylating them at specific regulatory sites, activating some and inhibiting others to
coordinate their activities.
The cell cycle is divided into four major phases. During the G1 phase cells synthesize RNAs and proteins,
during the S (synthesis) phase preparing for DNA synthesis and chromosome replication. After
progressing through the G2 phase, cells begin the complicated process of mitosis, also called the M
(mitotic) phase, which is divided into several stages. G1, S, and G2 together are called interphase.
Mitosis is conventionally broken down into four stages: prophase, metaphase, anaphase, and telophase.
Prophase: The chromosomes become condense during the prophase period of mitosis. Thus each
chromosome is composed of the two daughter DNA molecules resulting from DNA replication plus
the histones and other chromosomal proteins associated with them. The identical daughter DNA
molecules and associated chromosomal proteins that form one chromosome are referred to as
sister chromatids. Sister chromatids are attached to each other by protein cross-links along their
lengths. They become confined to a single region of association called the centromere.
Interphase: During interphase outer nuclear membrane is continuous with the endoplasmic
reticulum. In the prophase the cellular microtubules disassemble and reassemble into the mitotic
apparatus consisting of a football-shaped bundle of microtubules (the spindle) with a star-shaped
cluster of microtubules radiating from each end, or spindle pole.
Metaphase: During the metaphase period of mitosis, a multiprotein complex, the kinetochore,
assembles at each centromere. The kinetochores of sister chromatids then associate with
microtubules coming from opposite spindle poles.
Anaphase: During the anaphase period of mitosis, sister chromatids separate. They initially are
pulled by motor proteins along the spindle microtubules toward the opposite poles and then are
further separated as the mitotic spindle elongates.
Telophase: The physical division of the cytoplasm, called cytokinesis, and then yields two daughter
cells. Following mitosis, cycling cells enter the G1 phase, embarking on another turn of the cycle.
The cyclins are the regulatory subunits of the heterodimeric protein kinases that control cell-cycle
events. The concentration of cyclins is increase and decrease as cells progress through the cell cycle. The
catalytic subunits of these kinases, called cyclin-dependent kinases (CDKs), have no kinase activity unless
they are associated with a cyclin. Each CDK can associate with different cyclins, and the associated cyclin
determines which proteins are phosphorylated by a particular cyclin-CDK complex. There are three
major classes of cyclin-CDK complexes that control passage through the cell cycle: the G1, S-phase and
mitotic cyclin-CDK complexes.
When cells are stimulated to replicate, G1 cyclin-CDK complexes are expressed first. These prepare the
cell for the S phase by activating transcription factors that promote transcription of genes encoding
enzymes required for DNA synthesis and the genes encoding S-phase cyclins and CDKs.
The activity of S-phase cyclin-CDK complexes is initially held in check by inhibitors. Late in G1, the G1
cyclin-CDK complexes induce degradation of the S-phase inhibitors by phosphorylating them and
consequently stimulating their polyubiquitination by the multiprotein SCF ubiquitin ligase. Subsequent
degradation of the polyubiquitinated S-phase inhibitor by proteasomes releases active S-phase cyclin-
Mitotic cyclin-CDK complexes are synthesized during the S phase and G2, but their activities are held in
check by phosphorylation at inhibitory sites until DNA synthesis is completed. Once activated by
dephosphorylation of the inhibitory sites, mitotic cyclin-CDK complexes phosphorylated multiple
proteins that promote chromosome condensation, retraction of the nuclear envelope, assembly of the
mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate.
During mitosis, the anaphase promoting complex (APC), a multi subunit ubiquitin ligase,
polyubiquitinates key regulatory proteins marking them for proteasomal degradation. One important
substrate of the APC is securin, a protein that inhibits degradation of the cross-linking proteins between
sister chromatids. The polyubiquitination of securin by the APC is inhibited until the kinetochores
assembled at the centromeres of all chromosomes have become attached to spindle microtubules,
causing chromosomes to align at the metaphase plate.
Once all the chromosomes are aligned, the APC polyubiquitinates securin, leading to its proteasomal
degradation and the subsequent degradation of the cross-linking proteins connecting sister chromatids.
This sequence of events initiates anaphase by freeing sister chromatids to segregate to opposite spindle
poles.
Late in anaphase, the APC also directs polyubiquitination and subsequent proteasomal degradation of
the mitotic cyclins. Polyubiquitination of the mitotic cyclins by APC is inhibited until the segregating
chromosomes have reached the proper location in the dividing cell.
Degradation of the mitotic cyclins leads to inactivation of the protein kinase activity of the mitotic CDKs.
The resulting decrease in mitotic CDK activity permits constitutively active protein phosphatases to
remove the phosphates that were added to specific proteins by the mitotic cyclin- CDK complexes.
As a result, the now separated chromosomes decondense, the nuclear envelope re-forms around
daughter-cell nuclei, and the Golgi apparatus reassembles during telophase; finally, the cytoplasm
divides at cytokinesis, yielding the two daughter cells.
Passage through three critical cell-cycle transitions— G1 →S phase, metaphase →anaphase, and
anaphase → telophase and cytokinesis—is irreversible because these transitions are triggered by the
regulated degradation of proteins, an irreversible process. As a consequence, cells are forced to traverse
the cell cycle in one direction only.
The unreplicated-DNA checkpoint (1) prevents activation of cyclin A-CDK1 and cyclin B-CDK1 (i.e.,
mitosis promoting factor, MPF) by activation of an ATR-Chk1 protein kinase cascade that phosphorylates
and inactivates Cdc25C, thereby inhibiting entry into mitosis.
In the spindle-assembly checkpoint (2), Mad2 and other proteins inhibit activation of the APC specificity
factor (Cdc20) required for polyubiquitination of securin, thereby preventing entry into anaphase.
The chromosome-segregation checkpoint (3) prevents release of the Cdc14 phosphatase from nucleoli,
thereby blocking activation of the APC specificity factor (Cdh1) required for polyubiquitination of B-type
cyclins as well as induction of Sic1. As a result, the decrease in MPF activity required for the events of
telophase does not occur.
The integrin family comprises heterodimeric integral membrane proteins that function as adhesion
receptors, mediating many cell–matrix interactions. In vertebrates, at least 24 integrin heterodimers,
composed of 18 types of alpha subunits and 8 types of beta subunits in various combinations, are
known. A single beta chain can interact with any one of multiple alpha chains, forming integrins that
bind different ligands. Parts of both the alpha and the beta subunits of an integrin molecule contribute
to the primary extracellular ligand binding site. Ligand binding to integrins also requires the
simultaneous binding of divalent cations (positively charged ions).
The members of this large family have a remarkable ability to transmit signals in both directions across
the cell membrane. The binding of a matrix component to an integrin can send a message into the
interior of the cell, and conditions in the cell interior can send a signal outward to control binding of the
integrin to matrix.
Functions:
In epithelial cells integrins plays a major role in adhering cells to matrix in the underlying basal
lamina.
Some integrins, particularly those expressed by certain blood cells, participate in heterophilic cell–
cell interactions.
The members of this large family play important roles in adhesion and signaling in both epithelial
and nonepithelial tissues.
In addition to their adhesion function, integrins can mediate outside-in and inside-out transfer of
information (signaling). Integrin-mediated signaling pathways influence processes as diverse as cell
survival, cell proliferation, and programmed cell death.
Cell–Matrix Adhesion Is Modulated by Changes in the Binding Activity and Numbers of Integrins
Collagen
The collagens, a family of fibrous proteins found in all multicellular animals. Collagens are secreted in
large quantities by connective tissue cells and in smaller quantities by many other cell types. As a major
component of skin and bone, they are the most abundant proteins in mammals, where they constitute
2svo of the total protein mass.
The primary feature of a typical collagen molecule is its long, stiff, triple stranded helical structure, in
which three collagen polypeptide chains, called alpha chains, are wound around one another in a
ropelike superhelix. Collagens are extremely rich in proline and glycine, both of which are important in
the formation of the triple-stranded helix. Proline, because of its ring structure, stabilizes the helical
conformation in each alpha chain, while glycine is regularly spaced at every third residue throughout the
central region of the alpha chain. Being the smallest amino acid (having only a hydrogen atom as a side
chain), glycine allows the three helical cx chains to pack tightly together to form the final collagen
superhelix.
The human genome contains 42 distinct genes coding for different collagen alpha chains. Different
combinations of these genes are expressed in different tissues. Although in principle thousands of types
of triple-stranded collagen molecules could be assembled from various combinations of the 42 o chains,
only a limited number of triple-helical combinations are possible, and less than 40 types of collagen
molecules have been found.
Type I is by far the most common, being the principal collagen of skin and bone. It belongs to the class of
fibrillar collagens, or fibril-forming collagens, which have long ropelike structures with few or no
interruptions. After being secreted into the extracellular space, these collagen molecules assemble into
higher-order polymers called collagen fibrils, which are thin structures. Collagen fibrils often aggregate
into larger, cable like bundles, several micrometers in diameter called as collagen fibers.
Collagen types IX and XII are called fibril-associated collagens because they decorate the surface of
collagen fibrils. They are thought to link these fibrils to one another and to other components in the
extracellular matrix.
Type IV is a network-forming collagen, forming a major part of basal laminae, while type VII molecules
form dimers that assemble into specialized structures called anchoring fibrils. Anchoring fibrils help
attach the basal lamina of multilayered epithelia to the underlying connective tissue and therefore are
especially abundant in the skin.
The unique properties of each type of collagen are due mainly to differences in (1) the number and
lengths of the collagenous, triple-helical segments; (2) the segments that flank or interrupt the triple-
helical segments and that fold into other kinds of three-dimensional structures; and (3) the covalent
modification of the alpha chains.
Functions:
Collagen fibers, which provide mechanical strength and resilience
In scuruy, the often fatal disease caused by a dietary deficiency of vitamin c that was common in
sailors until the nineteenth century, the defective pro-alpha chains that are synthesized fail to form
a stable triple helix and are immediately degraded within the cell, and synthesis of new collagen is
inhibited. In healthy tissues, collagen is continually degraded and replaced (with a turnover time of
months or years, depending on the tissue). In scurvy, replacement fails, and within a few months,
with the gradual loss of the preexisting normal collagen in the matrix, blood vessels become fragile,
teeth become loose in their sockets, and wounds cease to heal.
Many vertebrate tissues, such as skin, blood vessels, and lungs, need to be both strong and elastic in
order to function. A network of elastic fibers in the extracellular matrix of these tissues gives them the
required resilience so that they can recoil after transient stretch. Elastic fibers are at least five times
more extensible than a rubber band of the same cross-sectional area.
Long, inelastic collagen fibrils are interwoven with the elastic fibers to limit the extent of stretching and
prevent the tissue from tearing. The main component of elastic fibers is elastin, a highly hydrophobic
protein (about 750 amino acids long), which, like collagen, is unusually rich in proline and glycine but,
unlike collagen, is not glycosylated and contains some hydroxyproline but no hydroxylysine. The elastin
Functions:
Elastin is the dominant extracellular matrix protein in arteries, comprising 50% of the dry weight of
the largest artery-the aorta. Mutations in the elastin gene causing a deficiency of the protein in mice
or humans result in narrowing of the aorta or other arteries and excessive proliferation of smooth
muscle cells in the arterial wall. Apparently, the normal elasticity of an artery is required to restrain
the proliferation of these cells.
They help to hold the lens in its place in the eye. In affected animals cause the displacement of the
lens.
Mutations in the fibrillin gene result in Marfan's syndrome, a relatively common human genetic
disorder.
Affected individuals are often unusually tall and lanky and have abnormalities of the skeleton and
joints.
It is a large glycoprotein found in all vertebrates and important for many cell-matrix interactions.
Fibronectin is a dimer composed of two very large subunits joined by disulfide bonds at one end. Each
subunit is folded into a series of functionally distinct domains separated by regions of flexible
polypeptide chain. The domains in turn consist of smaller modules, each of which are serially repeated
and usually encoded by a separate exon, suggesting that the fibronectin gene, like the collagen genes,
evolved by multiple exon duplications.
In the human genome, there is only one fibronectin gene, containing about 50 exons of similar size, but
the transcripts can be spliced in different ways to produce many different fibronectin isoforms. Sites in
Functions:
The mutant mice that are unable to make fibronectin die early in embryogenesis because their
endothelial cells are fail to form proper blood vessels. The defect is thought to result from
abnormalities in the interactions of these cells with the surrounding extracellular matrix, which
normally contains fibronectin.
Because the cell wall surrounding a plant cell prevents the cell from expanding, its structure must be
loosened when the cell grows. The amount, type, and direction of plant cell growth are regulated by
small-molecule hormones (e.g., indoleacetic acid) called auxins. The auxin-induced weakening of the cell
wall permits the expansion of the intracellular vacuole by uptake of water, leading to elongation of the
cell.
The plant hormone auxin, which is generally indole-3-acetic acid, binds to receptor proteins in the
nucleus. It helps plants grow toward light, grow upward rather than branch out, and grow their roots
down. It also regulates organ initiation and positioning and helps plants flower and bear fruit. Like
ethylene, it influences gene expression by controlling the degradation of gene regulatory proteins in the
nucleus, but instead of blocking the ubiquitylation and degradation of gene regulatory proteins required
for the expression of auxin-responsive genes, it stimulates the ubiquitylation and degradation of
repressor proteins that block the transcription of these genes in unstimulated cells.
Unlike the animal extracellular matrix, which is rich in protein and other nitrogen-containing polymers,
the plant cell wall is made almost entirely of polymers that contain no nitrogen, including cellulose and
lignin.
Synthesis:
Cellulose microfibrils are synthesized on the exoplasmic face of the plasma membrane from UDP glucose
and ADP-glucose formed in the cytosol. The polymerizing enzyme, called cellulose synthase, moves
within the plane of the plasma membrane as cellulose is formed, in directions determined by the
underlying microtubule cytoskeleton. Each enzyme complex has the six fold symmetry and contains the
protein product of three cellulose synthase (CESA) genes. Each CESA protein is essential for the
production of a cellulose microfibril. Three CESA genes are required for primary cell wall synthesis and
different three for secondary wall synthesis. As they being synthesized the nascent cellulose chains
assembly into the microfibrils. These are span out on the extra cellular membrane and form a layer.
Each new lamella forms internally to the previous one, so that the wall consists of concentrically
arranged lamellae, with the oldest on the outside. The most recently deposited microfibrils in elongating
cells commonly lie perpendicular to the axis of cell elongation.
Orientation:
For a plant cell to grow or change its shape, the cell wall has to stretch or deform. Because of their
crystalline structure, however, individual cellulose microfibrils are unable to stretch. Thus, stretching or
deformation of the cell wall must involve either the sliding of microfibrils past one another, the
separation of adjacent microfibrils, or both. The direction in which the growing cell enlarges depends in
part on the orientation of the cellulose microfibrils in the primary wall, which in turn depends on the
orientation of microtubules in the underlying cell cortex at the time the wall was deposited. The final
shape of a growing plant cell, and hence the final form of the plant, is determined by controlled cell
expansion. Expansion occurs in response to turgor pressure in a direction that depends in part on the
arrangement of the cellulose microfibrils in the wall.
If the entire system of cortical microtubules is disassembled by treating a plant tissue with a
microtubule-depolymerizing drug, the consequences for subsequent cellulose deposition are not as
straightforward as might be expected. The drug treatment has no effect on the production of new
cellulose microfibrils, and in some cases cells can continue to deposit new microfibrils in the preexisting
orientation. Any developmental change in the microfibril pattern that would normally occur between
successive lamellae, however, is invariably blocked. It seems that a preexisting orientation of microfibrils
can be propagated even in the absence of microtubules, but any change in the deposition of cellulose
microfibrils requires that intact microtubules be present to determine the new orientation.
Plant cells can change their direction of expansion by a sudden change in the orientation of their cortical
array of microtubules. Because plant cells cannot move (being constrained by their walls), the entire
morphology of a multicellular plant depends on the coordinated, highly patterned control of cortical
There are following model which put forward time to time to explain structure of plasma membrane:
Danielli and Davson (1935) proposed a “Trilamellar model”. According to this, the plasma membrane
is formed of a bimolecular layer of phospholipids (35 Å thick) sandwiched between two layers of
proteins (each 20 Å thick). Thus, the total thickness of plasma membrane, as per their model, should
be 20 Å + 35 Å + 20 Å = 75 Å (i.e., about 75 Å).
The model was proposed even before the plasma membrane was seen under the electron
microscope.
J.D. Robertson (1959) proposed a “unit membrane concept”. According to this, all biological
membranes shared the same basic structure:
(b) These have a characteristics trilaminar appearance when viewed with electron microscope.
(c) The three layers are a result of the same arrangement of proteins and lipids as proposed by
Danielli and Davson.
Singer and Nicolson (1972) put forward the “fluid mosaic model” of membrane structure. It is the
latest and most widely accepted model. According to this model, the cell membrane consists of a
highly viscous fluid matrix of two layers of phospholipids molecules. These serve as a relatively
impermeable barrier to the passage of most water soluble molecules. Protein molecules on their
complexes occur in the membrane, but not in continuous layer; instead, these occur as separate
particles asymmetrically arranged in a mosaic pattern. Some of these (peripheral or extrinsic
proteins) are loosely bound at the polar surfaces of lipid layers. Others (called integral or intrinsic
proteins), penetrate deeply into the lipid layer. Some of the integral proteins penetrate through the
phospholipids layers and project on both the surfaces. These are called Trans membranes or tunnel
proteins.
Passive Diffusion
Lipophilic solutes cross the membrane freely by dissolving in the lipid bilayer. This is passive diffusion.
Examples: ethanol (alcohol, contains both polar and non-polar regions); also fatty acids, glycerol,
steroids, etc. Also nonpolar gases like O=O (O2).
Polar or ionic small solutes may be transported across membranes if specific protein carriers are in the
membrane. Examples: sugars, amino acids, ions. Many substances cannot cross the membrane.
Examples: large molecules such as proteins, nucleic acids. Also small polar molecules or ions for which
there is no protein carrier.
• Glucose transporters- 5 different GLUT proteins and 2 types that cotransport Na and glucose (these
are used for secondary active transport)
Active transport
Membrane has specific protein carrier, also a requirement for energy (ATP or other form of energy). Will
move solute against a concentration gradient, so can concentrate material even if diffusion would favor
opposite direction of flow.
Example: Na+- K+ ATPase in nerve cells. Pumps Na+ to outside, K+ in, maintains electrical potential
against diffusion. When nerve cell “fires” the momentary gates open to let diffusion occur. Then pumps
are turned back on to restore potential.
There are mechanisms for moving larger molecules, but they don’t enter into cytoplasm.
Exocytosis
Membrane vesicle fuses with cell membrane, releases enclosed material to extracellular space. Ex:
release of digestive enzymes from pancreatic cells; mucus, milk, hormones, etc.
Endocytosis
Cell membrane invaginates, pinches in, and creates vesicle enclosing contents. Three common
situations:
Typically works on debris, bacteria, and other particulate matter. Contents of the “phagosome” are
usually fused with lysosome to create “phagolysosome”, where material is broken down. This is
especially common in white blood cells such as macrophages and other leukocytes.
Pinocytosis
Similar to Phagocytosis, but ingests fluid rather than particulate matter called “Cell drinking”. Ex. cells
lining blood capillaries take fluid from blood (but not red cells), move fluid across their cytoplasm,
release into extracellular space surrounding cells outside the capillary.
It is a very specialized system. Certain important molecules or ions are not brought into cell by transport
processes, but by CME. The body uses hormones to regulate membrane transport in this way Eg. 1. Iron
is carried through blood tightly bound to transferrin protein carrier. To get iron into cells, cell membrane
contains special receptor proteins that bind transferrin, move towards special regions of membrane
under which lie clathrin proteins. Endocytosis occurs inside clathrin “cage”, moves inside cell. Cage
eventually recycles back to cell surface, returning transferrin proteins to cell exterior. However, iron is
released inside cell, exits from vesicles, and becomes bound to ferritin.
The phospholipids bilayer, the basic structural unit of biomembranes, is essentially impermeable to
most water soluble molecules, ions, and water itself.
A very few molecules and no ions can cross a pure phospholipid bilayer at appreciable rates by passive
diffusion. Thus transport of most molecules into and out of cells requires the assistance of specialized
membrane proteins. Even transport of molecules with a relatively large partition coefficient (e.g., water
and urea) is frequently accelerated by specific proteins because their transport by passive diffusion
usually is not sufficiently rapid to meet cellular needs.
ATP-powered pumps
ATP-powered pumps (or simply pumps) are ATPases that use the energy of ATP hydrolysis to move ions
or small molecules across a membrane against a chemical concentration gradient or electric potential or
both. This process, referred to as active transport. In this case, transport of ions or small molecules
“uphill” against an electrochemical gradient, which requires energy, is coupled to the hydrolysis of ATP,
which releases energy. The overall reaction—ATP hydrolysis and the “uphill” movement of ions or small
molecules— is energetically favorable.
Channel proteins transport water or specific types of ions and hydrophilic small molecules down their
concentration or electric potential gradients. Such protein-assisted transport sometimes is referred to as
facilitated diffusion. Channel proteins form a hydrophilic passageway across the membrane through
which multiple water molecules or ions move simultaneously, single file at a very rapid rate. Some ion
channels are open much of the time; these are referred to as nongated channels. Most ion channels,
however, open only in response to specific chemical or electrical signals; these are referred to as gated
channels.
Transporters
Transporters (also called carriers) move a wide variety of ions and molecules across cell membranes.
Three types of transporters have been identified. Uniporters transport a single type of molecule down
its concentration gradient via facilitated diffusion. Glucose and amino acids cross the plasma membrane
into most mammalian cells with the aid of uniporters. In contrast, antiporters and symporters couple
the movement of one type of ion or molecule against its concentration gradient with the movement of
one or more different ions down its concentration gradient. These proteins often are called
cotransporters, referring to their ability to transport two different solutes simultaneously. Like ATP
pumps, cotransporters mediate coupled reactions in which an energetically unfavorable reaction (i.e.,
uphill movement of molecules) is coupled to an energetically favorable reaction. Note, however, that
the nature of the energy-supplying reaction driving active transport by these two classes of proteins
differs. ATP pumps use energy from hydrolysis of ATP, whereas cotransporters use the energy stored in
an electro chemical gradient. This latter process sometimes is referred to as secondary active transport.
The protein-mediated movement of glucose and other small hydrophilic molecules across a membrane,
known as uniport transport, exhibits the following distinguishing properties:
The rate of facilitated diffusion by uniporters is far higher than passive diffusion through a pure
phospholipid bilayer.
Because the transported molecules never enter the hydrophobic core of the phospholipid bilayer,
the partition coefficient K is irrelevant.
Transport occurs via a limited number of uniporter molecules, rather than throughout the
phospholipid bilayer. Consequently, there is a maximum transport rate Vmax that is achieved when
the concentration gradient across the membrane is very large and each uniporter is working at its
maximal rate.
Transport is specific. Each uniporter transports only a single species of molecule or a single group of
closely related molecules. A measure of the affinity of a transporter for its substrate is Km, which is
the concentration of substrate at which transport is half-maximal.
ATP-Powered Pumps
They have binding sites for ATP located on the cytosolic face of the membrane. There are four classes of
ATP-powered pumps. The members of three classes (P, F, and V) transport ions only, whereas members
of the ABC superfamily primarily transport small molecules.
All P-class ion pumps possess two identical catalytic subunits that contain an ATP-binding site. Most also
have two smaller subunits that usually have regulatory functions.
During the transport process, at least one of the subunits is phosphorylated (hence the name “P” class),
and the transported ions move through the phosphorylated subunit. This class includes the Na+/K+
ATPase in the plasma membrane, which maintains the low cytosolic Na+ and high cytosolic K+
concentrations typical of animal cells.
Another member of the P class, found in acid-secreting cells of the mammalian stomach, transports
protons (H+ ions) out of and K+ ions into the cell.
The structures of F-class and V-class ion pumps are similar to one another but unrelated to and more
complicated than P-class pumps. F- and V-class pumps contain several different transmembrane and
cytosolic subunits. All V and F pumps transport only protons and does not involve a phosphoprotein
intermediate. V-class pumps generally function to maintain the low pH of acidic vesicles in animal cells
F-class pumps are found in bacterial plasma membranes and in mitochondria and chloroplasts. V pumps
generally function to power the synthesis of ATP from ADP and Pi by movement of protons from the
exoplasmic to the cytosolic face of the membrane down the proton electrochemical gradient. Because
of their importance in ATP synthesis in chloroplasts and mitochondria, F-class proton pumps, commonly
called ATP synthases.
In ABC superfamily each ABC protein is specific for a single substrate or group of related substrates,
which may be ions, sugars, amino acids, phospholipids, peptides, polysaccharides, or even proteins. All
ABC transport proteins share a structural organization consisting of four “core” domains: two
transmembrane (T) domains, forming the passageway through which transported molecules cross the
membrane, and two cytosolic ATP-binding (A) domains.
In skeletal muscle cells, Ca2+ ions are concentrated and stored in the sarcoplasmic reticulum (SR);
release of stored Ca2+ ions from the SR lumen into the cytosol causes contraction, as discussed in
Chapter 19. A P-class Ca2+ ATPase located in the SR membrane of skeletal muscle pumps Ca2+from the
cytosol into the lumen of the SR, thereby inducing muscle relaxation. The current model for the
mechanism of action of the
Ca2+ ATPase in the SR membrane involves two conformational states of the protein termed E1 and E2.
Coupling of ATP hydrolysis with ion pumping involves several steps that must occur in a defined order.
A second important P-class ion pump present in the plasma membrane of all animal cells is the Na+/K+
ATPase. This ion pump is a tetramer of subunit composition alpha2 beta2. The small, glycosylated beta
polypeptide helps newly synthesized alpha subunits to fold properly in the endoplasmic reticulum but
apparently is not involved directly in ion pumping. The mechanism of action of the Na+/K+ ATPase is
similar to that of the muscle calcium pump, except that ions are pumped in both directions across the
membrane.
All V-class ATPases transport only H+ ions. The V-class proton pumps contain two discrete domains: a
cytosolic hydrophilic domain (V1) and a transmembrane domain (V0) with multiple subunits in each
domain. Binding and hydrolysis of ATP by the B subunits in V1 provide the energy for pumping of H+ ions
through the proton-conducting channel formed by the c and a subunits in V0. By themselves ATP-
powered proton pumps cannot acidify the lumen of an organelle (or the extracellular space) because
these pumps are electrogenic; that is, a net movement of electric charge occurs during transport.
Pumping of just a few protons causes a buildup of positively charged H+ ions on the exoplasmic (inside)
face of the organelle membrane. For each H+ pumped across, a negative ion (e.g., OH- or Cl-) will be
“left behind” on the cytosolic face, causing a buildup of negatively charged ions there. These oppositely
charged ions attract each other on opposite faces of the membrane, generating a charge separation, or
electric potential, across the membrane. As more and more protons are pumped, the excess of positive
charges on the exoplasmic face repels other H+ ions, soon preventing pumping of additional protons
long before a significant transmembrane H+ concentration gradient had been established In order for an
organelle lumen or an extracellular space (e.g., the lumen of the stomach) to become acidic, movement
of protons must be accompanied either by (1) movement of an equal number of anions (e.g., Cl-) in the
same direction or by (2) movement of equal numbers of a different cation in the opposite direction. The
first process occurs in lysosomes and plant vacuoles whose membranes contain V-class H+ ATPases and
anion channels through which accompanying Cl- ions move. The second process occurs in the lining of
the stomach, which contains a P-class H+/K+ ATPase that is not electrogenic and pumps one H+ outward
and one K+ inward.
The ABC superfamily of transport proteins contain two transmembrane (T) Domains and two cytosolic
ATP-binding (A) domains. The T domains, each built of six membrane spanning alpha helices, form the
pathway through which the transported substance (substrate) crosses the membrane and determine
the substrate specificity of each ABC protein. The plasma membrane of many bacteria contains
numerous permeases that belong to the ABC superfamily. These proteins use the energy released by
hydrolysis of ATP to transport specific amino acids, sugars, vitamins, or even peptides into the cell.
The tumor cells have resistance to several drugs with unrelated chemical structures. Such cells
eventually were shown to express elevated levels of a multidrug-resistance (MDR) transport protein
known as MDR1. This protein uses the energy derived from ATP hydrolysis to export a large variety of
drugs from the cytosol to the extracellular medium. The Mdr1 gene is frequently amplified in multidrug-
resistant cells, resulting in a large overproduction of the MDR1 protein. This transport is described by
the flippase model. According to this model, MDR1 “flips” a charged substrate molecule from the
cytosolic to the exoplasmic leaflet, an energetically unfavorable reaction powered by the coupled
ATPase activity of the protein. Following steps are involved in flippase model transport:-
Step 1: The hydrophobic portion (black) of a substrate molecule moves spontaneously from the cytosol
into the cytosolic-facing leaflet of the lipid bilayer, while the charged end (red) remains in the cytosol.
Step 2: The substrate diffuses laterally until encountering and binding to a site on the MDR1 protein
within the bilayer.
Step 3: The protein then “flips” the charged substrate molecule into the exoplasmic leaflet, an
energetically unfavorable reaction powered by the coupled hydrolysis of ATP by the cytosolic domain.
Steps 4 and 5: Once in the exoplasmic face, the substrate again can diffuse laterally in the membrane
and ultimately moves into the aqueous phase on the outside of the cell.
The plasma membranes of animal cells contain many open K+ channels but few open Na2+, Cl-, or Ca2-
channels. As a result, the major ionic movement across the plasma membrane is that of K+ from the
inside outward, powered by the K+ concentration gradient, leaving an excess of negative charge on the
inside and creating an excess of positive charge on the outside. Thus the outward flow of K+ ions
through these channels, called resting K+ channels, is the major determinant of the inside-negative
membrane potential. Like all channels, these alternate between an open and a closed state, but since
their opening and closing are not affected by the membrane potential or by small signaling molecules,
these channels are called nongated. All ion channels exhibit specificity for particular ions: K+ channels
allow K+ but not closely related Na+ ions to enter, whereas Na+ channels admit Na+ but not K+.
The ability of the ion-selectivity filter in K+ channels to select K+ over Na+ is due mainly to backbone
carbonyl oxygens on glycine residues located in a Gly-Tyr-Gly sequence that is found in an analogous
position in the P segment in every known K+ channel. As a K+ ion enters the narrow selectivity filter, it
loses its water of hydration but becomes bound to eight backbone carbonyl oxygens as a result
relatively low activation energy is required for passage of K+ ions through the channel. Because a
dehydrated Na+ ion is too small to bind to all eight carbonyl oxygens that line the selectivity filter, the
activation energy for passage of Na+ ions is relatively high. This difference in activation energies favors
passage of K+ ions over Na+ by a factor of thousand. Calcium ions are too large to pass through a K+
channel with or without their bound water.
When the transported molecule and cotransported ion move in the same direction, the process is called
symport; when they move in opposite directions, the process is called antiport. Some cotransporters
transport only positive ions (cations), while others transport only negative ions (anions). An important
example of a cation cotransporter is the Na+/H+ antiporter, which exports H+ from cells coupled to the
energetically favorable import of Na+. An example of an anion cotransporter is the AE1 anion antiporter
protein, which catalyzes the one-for-one exchange of Cl- and HCO3- across the plasma membrane.
Transepithelial Transport
The cell junctions are the specialized regions of the plasma membrane that connect epithelial cells
forming the sheet like lining of the intestines and other body surfaces. The absorption of nutrients from
the intestinal lumen occurs by the import of molecules on the luminal side of intestinal epithelial cells
and their export on the blood- facing side by a two-stage process called transcellular transport.
State 1:- In the closed, resting state, the voltage-sensing alpha helices, which have positively charged
side chains every third residue, are attracted to the negative charges on the cytosolic side of the resting
membrane. This keeps the gate segment in a position that blocks the channel, preventing entry of Na+
ions.
State 2:- In response to a small depolarization the voltage-sensing helices rotate in a screw like manner
toward the outer membrane surface causing an immediate conformational change in the gate segment
that opens the channel.
State 3:- The voltage-sensing helices rapidly return to the resting position and the channel-inactivating
segment moves into the open channel, preventing passage of further ions.
State 4:- Once the membrane is repolarized, the channel-inactivating segment is displaced from the
channel opening and the gate closes; the protein reverts to the closed, resting state and can be opened
again by depolarization.
Each subunit contains an M2 alpha helix (red) that faces the central pore. Aspartate and glutamate side
chains at both ends of the M2 helices form two rings of negative charges that help exclude anions from
and attract cations to the channel. The gate, which is opened by binding of acetylcholine, lies within the
pore. (b) Cross section of the exoplasmic face of the receptor showing the arrangement of subunits
around the central pore. The two acetylcholine binding sites are located about 3 nm from the
membrane surface.
Electric character of ion transport may be electroneutral i.e. electrically silent either by symport of the
oppositely charged ions or antiport of similarly charged ions, or electrogenic i.e. results in charge
separation across the membrane. Electrogenic transport affect can be affected by membrane potential.
For example the Na+/K+ pump import 2K+ and simultaneously exports 3Na+; that is, it moves 1 positive
charge out of the cell. Its electrogenic operation directly contribute to the negative inside membrane
potential, which is evidenced by the fact that stopping the pump using an alkaloid inhibitor oubain
causes an immediate slight depolarization of the cell membrane.
Electric potential across cell membrane are a function of the electrolyte concentrations in the
intracellular and extracellular solutions and of selective permiabilities of the ions. Active transport of
ions by ATP driven ion pumps generate and maintain ionic gradient. In addition to ion pumps which
transport ions against concentration gradients, plasma membrane contains channel protein that allows
ions to move through it at different rates down their concentration gradient. Ion concentration
gradients and selective movements of ions create a difference in electric potential or voltage across
plasma membrane. This is called membrane potential. Typically membrane potential is between -30 and
-70 mv (minus sign indicate that inside of cell always negative with respect to the outside). In nerve and
muscle cells membrane potential at resting state is called as the resting potential.
For many cells the principal diffusion ions are the sodium, potassium and chloride. The exact
quantitative expression for membrane potential is given by Goldman Hodgkin Katz equation which
relates the membrane potential to infra and extracellular concentration of these ions and their
permeability coefficient.
In most of the cells the resting membrane potential is dictated predominantly by the K+ gradient and its
value is not far from the K+ equilibrium potential. The reason for this is that at rest K+ is the most
permeable ion across the cell membrane. In contrast Na+ and Ca2+ ions are very far from their
equilibrium and they are interacted inward both by the negative membrane potential and by their
concentration gradient. However their contribution to the resting potential is very modest (in case of
the Ca2+ it is negligible) because at rest the Na+ and Ca2+ channels are closed and therefore membrane
permeability is very small for these ions.
In all animal cells the electric potential across the plasma membrane is generated by primarily by
movement of cytosolic K+ ions through the resting K+ ions channels to the external medium. Movement
of cytosolic K+ ions through the resting K+ ion channels to the external medium. Movement of K+ ions
across membrane down its concentration gradient leaves an excess negative charge on the cytosolic
face and deposited a positive charge on axoplasmic face. In nerve cells the resting potential is -70 mv
with the equilibrium potential and is largely due to much higher potassium than sodium permeability
across the membrane. In plants and fungi the membrane potential is maintained by the ATP driven
pumping of protons from the cytosol across the membrane.
A body cavity is any fluid filled space in a multicellular organism. However, the term usually refers to the
space, located between an animal’s outer covering (epidermis) and the outer lining of the gut cavity,
where internal organs develop. A coelom is a cavity lined by an epithelium derived from mesoderm.
Organs formed inside a coelom can freely move, grow, and develop independently of the body wall
while fluid cushions and protects them from shocks.
Arthropods and mollusks have a reduced (but still true) coelom. Their principal body cavity is the
hemocoel of an open circulatory system.
Mammalian embryos develop two coelomic cavities: the intraembryonic coelom and the
extraembryonic coelom (or chorionic cavity). The intraembryonic coelom is lined by somatic and
splanchnic lateral plate mesoderm, while the extraembryonic coelom is lined by extraembryonic
mesoderm. The intraembryonic coelom is the only cavity that persists in the mammal at term, which is
why its name is often contracted to simply coelomic cavity. Subdividing the coelomic cavity into
compartments, for example, the pericardial cavity / pericardium, where the heart develops, simplifies
discussion of the anatomies of complex animals.
Formation
Coelom formation begins in the gastrula stage. The developing digestive tube of an embryo forms as a
blind pouch called the archenteron.
In Protostomes the coelom forms by a process known as schizocoely. The archenteron initially forms,
and the mesoderm splits into two layers: the first attaches to the body wall or ectoderm, forming the
parietal layer and the second surrounds the endoderm forming the visceral layer or alimentary canal.
The space between the parietal layer and the visceral layer is known as the coelom or body cavity. In
Deuterostomes, the coelom forms by enterocoely: mesoderm buds from the walls of the archenteron
and hollows to become the coelomic cavities.
Origins
The origin of the coelom is uncertain. The oldest known animal to have had a body cavity is
Vernanimalcula.
The enterocoel theory says that the coelom evolved from gastric pouches of cnidarian ancestors.
This theory is supported by research on flatworms and small worms recently discovered in marine
fauna.
Coelomates
Also known as eucoelomates or "true coelom" have a fluid filled body cavity called a coelom with a
complete lining called peritoneum derived from mesoderm (one of the three primary tissue layers). The
complete mesoderm lining allows organs to be attached to each other so that they can be suspended in
a particular order while still being able to move freely within the cavity. Most bilateral animals, including
all the vertebrates, are Coelomates.
Pseudocoelomates
These animals have a pseudocoel (literally “false cavity”), which is a fully functional body cavity. Tissue
derived from mesoderm only partly lines the fluid filled body cavity of these animals. Thus, although
organs are held in place loosely, they are not as well organized as in a coelomate. All pseudocoelomates
are protostomes; however, not all protostomes are pseudocoelomates. An example of a
Pseudocoelomate is the roundworm. Pseudocoelomate animals are also referred to as Hemocoel and
Blastocoelomate.
In some protostomes, the embryonic blastocoele persists as a body cavity. These protostomes have a
fluid filled main body cavity unlined or partially lined with tissue derived from mesoderm. This fluid-filled
space surrounding the internal organs serves several functions like distribution of nutrients and removal
of waste or supporting the body as a hydrostatic skeleton.
A pseudocoelomate is any invertebrate animal with a three-layered body and a pseudocoel. The coelom
was apparently lost or reduced as a result of mutations in certain types of genes that affected early
development. Thus, pseudocoelomates evolved from Coelomates.
Important characteristics:
Lack a vascular blood system: Diffusion and osmosis circulate nutrients and waste products
throughout the body.
Lack a skeleton: Hydrostatic pressure gives the body a supportive framework that acts as a skeleton.
No segmentation
Body wall: Epidermis and muscle, often syncytial and usually covered by a secreted cuticle.
Most are microscopic
Parasites of almost every form of life (although some are free living)
Acoelomate
These animals, like flatworms, have no body cavity at all. Organs have direct contact with the
epithelium. Semi-solid mesodermal tissues between the gut and body wall hold their organs in place.
Organisms showing acoelomate formation include the platyhelminthes (flatworms, tapeworms etc.) The
coelom can be used for diffusion of gases and metabolites etc. These creatures do not have this need, as
the surface area to volume ratio is large enough to allow absorption of nutrients and gas exchange by
diffusion alone, due to dorso-ventral flattening.
The major distinctions between deuterostomes and protostomes are found in embryonic development.
In animals the embryo forms a dent on one side called as the blastopore which deepens to become the
archenteron which is the first phase in the growth of the gut. In deuterostomes the original dent
becomes the anus while the gut eventually tunnels through to make another opening which forms the
mouth. The protostomes were so named because it used to be thought that in their embryos the dent
formed the mouth while the anus was formed later, at the opening made by the other end of the gut.
More recent research, however, shows that in protostomes the edges of the dent close up in the
middle, leaving openings at the ends which become the mouth and anus. However, this idea has been
challenged, because the platyhelminthes have a single mouth which leads into a blind gut (with no
anus). The genes employed in the embryonic construction of this mouth are the same as those
expressed around the protostome mouth.
There are other significant differences between the protostome and deuterostome patterns of
development:
Most protostomes are schizocoelomates, meaning a solid mass of the embryonic mesoderm splits to
form a coelom. A few, such as Priapulids, have no coelom, but they may have descended from
schizocoelomate ancestors. On the other hand all known deuterostomes are enterocoelous,
meaning that the coelom is formed from longitudinal pouches of the archenteron which then
become separate cavities.
Within the Protostomes a number of phyla undergo what is known as spiral cleavage which is
determinate, meaning that the fate of the cells is determined as they are formed. This is in contrast
to deuterostomes which have radial cleavage that is indeterminate.
Cryopreservation derives from the Greek cryos, meaning "cold." It refers to the preservation of
biological tissue in sub-zero temperatures. Basic biological processes are therefore prevented, including
those that lead to cell death.
Fertility clinics commonly use cryopreservation on blastocysts (between two and eight cells) and semen
(if the prospective father is to undergo testicular surgery). Cryopreservation is also used on blood and
stem cells.
A risk to the tissue upon freezing is ice damage. If tissue is frozen untreated, ice would build up in the
cells, causing rupture of the cell membrane. Because freezing draws water out of cells, ice crystal
formation from without could also pierce or crush cell membranes.
For this reason, freezing is performed slowly to allow water to leave the cells. Glycerol was used to
replace extracellular water to avoid ice crystals. Today, dimethyl sulphoxide is used to prevent ice
formation.
Types of Cryopreservation
Cryopreservation is the storage of living organisms at an ultra-low temperature so that they can be
revived and restored at a later date. The length of time you wish to have the organisms frozen
determines the process. Ultra cold freezers can preserve living cells for weeks or even years; liquid
nitrogen is used for longer periods of time. Cryopreservation is most often used to aid reproduction
among humans.
Sperm Cryopreservation
Insemination using previously frozen sperm has a greater success rate than reversing a vasectomy or
extracting sperm directly from the testicle. Sperm cryopreservation is most often used before
undergoing cancer therapy that could cause infertility and before having prostate or testicular surgery.
Sperm cryopreservation is also used for men who work high-risk jobs with chemicals, have a low sperm
count or ejaculatory dysfunction.
Egg Cryopreservation
Egg cryopreservation is more difficult than sperm cryopreservation and involves a multistage lab
procedure. The eggs can later be thawed, fertilized, and implanted in order to achieve pregnancy. Egg
cryopreservation is most often used for professional women and single women who wish to have
children at a later date. Egg cryopreservation is used in women who have to undergo radiation,
chemotherapy, or have to have their ovaries removed.
Cryopreservation Techniques
Requirements
Cryoprotectants are used to help in the process of vitrification, a rapid freezing technique converting the
object into a glass-like state free of the formation of crystals. Cryoprotectants lower the freezing point
and prevent ice formation. The choice of the cryoprotectant agent is related to the type of cell or
organism being frozen. The agent is diluted to the strength necessary for the type of object. Some of the
cryoprotectants absorb quickly into the body through the skin and can deliver harmful chemicals into
the body. Care is necessary during this phase to reduce the likelihood of harmful exposure.
Rate of Cooling
The rate of cooling is important as this affects the formation of ice crystals. Programmable cell-freezing
equipment allows a selection of variable rates for the different portions. Specially designed containers
are used to accomplish the uniform rate of cooling. Various methods are used depending on the object
being frozen. Uncontrolled cooling is done by placing the containers in a -60-degree C freezer for 90
minutes. Semi-controlled cooling is also conducted in a -60-degree C freezer. Controlled cooling uses a
programmable cooling unit to cool the cells at 1 degree C per minute to -40 degrees C.
Thawing
The process of thawing is rapid, and is done by placing the container in a cold water bath with gentle
agitation during the warming phase to accelerate the thawing process while taking care not to damage
the matter therein. Transferring the material to a fresh growth medium will avoid any exposure to the
Cryopreservation is, simply put, flash freezing a biological tissue or organism in liquid nitrogen (N2). The
tissue may be a nonliving sample, or a living sample for purposes of revivification. This is the practice of
cryonics, and it sounds far simpler than it is. Only simple organisms stand up to cryopreservation
because these are more self-contained; so a sperm cell can be frozen and revivified, but thus far, a
human cannot.
Liquid Nitrogen
Nitrogen does not exist as a liquid; it exists as a gas and is plentiful in the air. Under tremendous
pressure, it becomes liquid nitrogen, the cold liquid. Doctors use it to freeze warts and skin lesions, and
then scrape them off while they are frozen. Nitrogen has two advantages as a freezing agent. First, it is
chemically fairly neutral, so it will not poison the life form that it preserves. Secondly, it is easily
manufactured and stored at its liquid temperatures of--196 degrees centigrade, or 321 degrees
Fahrenheit. Finally, it is quite cold; liquid oxygen is not nearly so cold, so is a poor freezing agent.
When it Works
The cryopreservation method works for simple, single-cell organisms, like sperm, ova, new embryos,
stem cells or plant seeds. The reason is that these are relatively noncomplex organisms of only one cell
or a few cells, or which exist en masse. Thus, only one or a few cells must revivify. For every frozen
sperm sample, thousands will perish, but thousands will live.
When it fails
More complex creatures, like a woolly mammoth, the Iceman of the Italian Alps, and baseball Hall-of-
Famer Ted Williams (whose head is cryogenically frozen), cannot be revivified. In order to do so, every
cell that makes up the complex mechanism of, for example, the finger; the nerve along the finger, and
the brain, must come to life. So far no one has achieved revivification of a complex creature (not even a
frog).
Cryonics
A few humans (including Ted Williams) have been cryogenically frozen. (Contrary to urban legend, Walt
Disney is not.) In these cases, the head is removed from the body and cryogenically frozen. In order to
revivify these people, several steps must be conquered:
Biological Samples
Not all cryopreservation is meant to revivify the subject; sometimes, a scientist wishes to preserve a
tissue sample (like a biopsy) for later study. Or, the scientist may wish to freeze a sample to take a
snapshot; when using a bioreactor to grow a particular type of cell, the scientist may wish to count the
cells in a given sample to determine the rate of reproduction. Some frozen blood samples dating back to
the 1950s revealed that AIDS has been present in Africa for several decades.
The cytoskeleton is unique to eukaryotic cells. It is a dynamic three-dimensional structure that fills the
cytoplasm. This structure acts as both muscle and skeleton, for movement and stability. The long fibers
of the cytoskeleton are polymers of subunits. The primary types of fibers comprising the cytoskeleton
are microfilaments, microtubules, and intermediate filaments.
Microfilaments
Microfilaments are fine, thread-like protein fibers, 3-6 nm in diameter. They are composed
predominantly of a contractile protein called actin, which is the most abundant cellular protein.
Microfilaments' association with the protein myosin is responsible for muscle contraction.
Microfilaments can also carry out cellular movements including gliding, contraction, and cytokinesis.
Actin Filaments
The actin subunit is a single globular polypeptide chain and is thus a monomer rather than a dimer. Each
actin filament has a binding site for ATP. The actin subunits are assembled head-to-tail to generate
filament with a distinct structural polarity. The actin filament can be considered to consist of two
parallel protofilaments that twist around each other in a right handed helix. The structural polarity of
actin filaments is created by the regular parallel orientation of all their subunits.
Myosin
Myosin is a motor protein. The form of myosin found in muscle and first identified was myosin-II. Each
myosin-II molecule is an oligomer composed of one pair of identical heavy chains and two pairs of non-
identical light chains.
Each of the heavy chain has a globular head domain at its N-terminus that contains an actin binding site
and a catalytic site that hydrolyzes ATP. Globular head domain is followed by a very long amino acid
sequence that forms an extended coil that mediate heavy chain dimerization. The two light chains bind
The long coiled tailed bundle itself with the tail of other myosin molecules. These tail-tail interactions
result in the formation of the large bipolar thick filament that have several hundred heads oriented in
opposite direction at the two end of the thick filament.
When myosin is treated with the protease trypsin it is cleaved into two fragments called light
meromyosin and heavy meromyosin. Light meromyosin consists of most of the alpha helical rode of the
myosin molecules. Heavy meromyosin consists of globular heads with light chain and the remaining rod
section. Subsequent treatment of heavy meromyosin with papain releases two globular protein heads
called S1 subfragment and a rod like section called S2 subfragment.
The myosin involves in transport of membrane vesicles and organelle along actin filament, phagocytosis
and extension of pseudopods in amoebae.
Microtubules
Microtubules are cylindrical tubes, 20-25 nm in diameter. They are composed of subunits of the protein
tubulin--these subunits are termed alpha and beta. Microtubules act as a scaffold to determine cell
shape, and provide a set of "tracks" for cell organelles and vesicles to move on. Microtubules also form
the spindle fibers for separating chromosomes during mitosis. When arranged in geometric patterns
inside flagella and cilia, they are used for locomotion.
The basic building block unit of microtubules is the tubulin that composed of two subunit the α-tubulin
and β-tubulin. A large number of additional cellular molecules called microtubule associated proteins
(MAP) bind to the surface of the microtubule and determine and control its many functions. MAPs play
critical role in the regulation of microtubule polymerization and dynamics, in the organisation of
microtubule arrays and in the functional interactions of the microtubules with other cellular
components.
Head-to-tail fashion. All of the β-tubulin subunits face one end of the microtubules and the α-tubulin
subunits face the opposite end. The ends are designated either plus or minus based upon their different
dynamic and structural properties. The β-subunits are exposed at the plus end while the α-subunits are
exposed at the minus ends. Each linear array of tubulin heterodimer is called a protofilament.
Microtubule consists of 13 protofilaments arranged into a tube structure.
Microtubule in a cell are usually organised at the microtubule organizing center (MTOC). In animal cells
MTOC contain a pair of centrioles and called as centromere. The minus ends are embedded at MTOC
and the plus end are away from it. One of the known proteins of the MTOC is a form of tubulin called ϒ-
tubulin which does not form microtubule itself and does not become incorporated into microtubule but
does appear to function in the nucleation of microtubule formation.
Single microtubule in cytosol is very common but many specialized microtubule arrays also exists in
which the microtubules are fused along their lengths into doublet or triplet microtubule structure.
Doublet microtubules consist of 10 or 11 protofilaments of second microtubule fused to entire 13
protofilaments first microtubules and triplet microtubules consist of an additional 10 or 11
protofilaments of third microtubule fused to second microtubules. Doublet microtubule occurs in the
long shaft of the motile cell appendages called cilia and flagella. Triplet microtubule occurs in the basal
bodies structure situated at the base of cilia and flagella and in centriole.
Lag Phase: The lag phase correspond to the time taken for nucleation.
Growth Phase: The growth phase occurs as monomers add to the exposed ends of the growing
filaments causing filaments elongation.
Steady Phase: The steady phase is reached when the growth of the filament due to monomer
addition is exactly balanced by the depolymerization from the ends. The number of monomer that
adds to the polymer per second depends upon the concentration of the free subunits. The
concentration of free subunits left at steady stage point is called as the critical concentration.
The rate of the subunit addition at the end of a filament is the product of the free subunit
concentration. The rate of polymerization as well as depolymerization is much faster for the plus end of
the filament than for the minus end. When the αβ-tubulin concentration is high than the critical
concentration at the plus end but lower than the critical concentration at the minus end, microtubule
can treadmill by adding subunits to one end and dissociating subunits from opposite end. This special
state in polymer dynamics is called as the treadmilling keeps the polymer length unchanged. A second
behaviour is called dynamic instability which is characterized by the phase transitions or switching
between phases of relatively slow growing and rapid shortening at the end of individual microtubule.
Dynein are the family of the minus end directed microtubule motor proteins. They are composed of two
or three heavy chains that include the motor domain and a large and a variable number of associated
light chains. Dynein family can be classified into two major groups:
Cytoplasmic dynein: They are found in eukaryotic cells and important for the vesicle targeting, as
well as for the localization of Golgi apparatus near the center of the cell.
Intermediate Filaments
Intermediate filaments are about 10 nm diameter and provide tensile strength for the cell.
Examples of the cytoskeleton in epithelial cells in the epithelial (skin) cells of the intestine, all three
types of fibers are present. Microfilaments project into the villi, giving shape to the cell surface.
Microtubules grow out of the centrosome to the cell periphery. Intermediate filaments connect adjacent
cells through desmosomes.
Intermediate filaments are the alpha helical rods that assemble into ropelike filament. Intermediate
filaments proteins are classified into four major categories based on their sequence and tissue
distribution:
Vimentin: Expressed in leukocytes, blood vessels, endothelial cells, some epithelial cells and
mesenchyme cells such as fibroblasts.
Desmin: Expressed in muscle cells responsible for the stabilizing sarcomere in contracting
muscles.
Glial Fibrillary Acidic Protein (GFAP): It forms the filaments in the glial cells that surround
neurons and in astrocyte.
Cilia are hair-like structures that can beat in synchrony causing the movement of unicellular
paramecium. Cilia are also found in specialized linings in eukaryotes. For example, cilia sweep fluids past
stationary cells in the lining of trachea and tubes of female oviduct.
Flagella are whip-like appendages that undulate to move cells. They are longer than cilia, but have
similar internal structures made of microtubules. Prokaryotic and eukaryotic flagella differ greatly.
Vesicle movement between organelles and the cell surface frequently studied in the squid axon.
Cytoplasmic streaming
Cell division--cytokinesis
Cellular motors Cells have protein motors that bind two molecules, and using ATP as energy, cause one
molecule to shift in relationship to the other. Two types of these protein motors are myosin and actin,
and dynein or kinesin and microtubules. These families of proteins all have a motor end, but may have
several kinds of different molecular structures on the binding end. When these proteins bind, they can
cause many different molecules, organelles, etc. to move. To the right is an example of the different
binding ends found in the kinesin family of motors.
When linked to other microtubules, protein motors can cause motion if the ends are fixed or extend the
lengths of the fiber bundles if the ends are free.
Broken motors in healthy individuals, the protein dystrophin is part of the linkage between the cellular
cytoskeleton and the adhesive proteins on the outside of the cell. In Duchenne Muscular Dystrophy the
gene that code for dystrophin is defective which resulting in muscle degeneration and finally death. This
disease is X-linked recessive and occurs in 1 out of every 3,500 males.
The spindle is a bundle of microtubules with associated proteins. It is divided into opposing halves at
the equator of the cell by the metaphase chromosomes.
An aster is a radial array of microtubules at each pole of the spindle. In each half of the spindle, a
single centrosome at the pole organizes three distinct sets of microtubules whose (-) ends all point
toward the centrosome.
One set of the astral microtubules originate forms the aster. They radiate outward from the
centrosome toward the cortex of the cell where they help position the mitotic apparatus and later
help to determine the cleavage plane in cytokinesis.
Polar microtubules do not interact with chromosomes but instead overlap with polar microtubules
from the opposite pole. Two types of interactions hold the spindle halves together to form the
bilaterally symmetric mitotic apparatus: (1) lateral interactions between the overlapping (+) ends of
the polar microtubules and (2) end-on interactions between the kinetochore microtubules and the
kinetochores of the sister chromatids.
The linkage of metaphase chromosomes to the (+) ends of kinetochore microtubules is mediated by
a large protein complex, the kinetochore, which has several functions: to trap and attach
microtubule ends to the chromosomes, to generate force to move chromosomes along
microtubules, and to regulate chromosome separation and translocation to the poles.
In an animal cell, the kinetochore forms at the centromere and is organized into an inner and outer
layer embedded within a fibrous corona. In all eukaryotes, three components participate in
attaching chromosomes to microtubules: the centromere, kinetochore and spindle proteins, and the
cell-cycle machinery.
At prophase, polar microtubules growing randomly from opposite poles are aligned with the aid of (-)
end–directed motors.
After alignment, (+) end–directed mitotic kinesins, including the bipolar kinesin BimC, generate pushing
forces that separate the poles.
In addition, a (-) end–directed force exerted by cytosolic dynein located at the cortex may pull asters
toward the poles. Similar forces act later at anaphase.
Some chromosomes first encounter the side, not the end, of a microtubule, interacting with the
microtubule through proteins at the kinetochore. Kinetochore-associated (+) end–directed motor
proteins (e.g., MCAK) then move the chromosome to the (+) end, thereby stabilizing the
microtubule.
Cytoplasmic dynein, a (-) end–directed motor at the kinetochore and a (+) end–directed motor at
the spindle pole pull chromosomes toward the pole
CENP-E, a kinesin that does not mediate movement, keeps the kinetochore tethered to the
kinetochore microtubule
Treadmilling of tubulin subunits briefly stabilizes the lengths of spindle microtubules at metaphase
by balancing assembly at the kinetochore with disassembly at the poles
A gene is a segment of nucleic acid that contains the information necessary to produce a functional
product, usually a protein. Genes consist of a long strand of DNA (RNA in some viruses) that contains a
promoter, which controls the activity of a gene, and a coding sequence, which determines what the
gene produces.
The genes are made up of a coding alphabet of 4 nucleotides made up of 4 bases:- Adenine(A), Thymine
(T), Guanine (G) and Cytosine (C) The bases Adenine(A) and Guanine(G) are Purines; while Thymine (T)
and Cytosine (C) are Pyrimidines.
Genes are poly-nucleotides. Each nucleotide is made up of a base group, a sugar and one ore phosphate
groups. The sugar group forms the backbone of DNA. The phosphate groups are responsible for linking
one nucleotide to another. The nitrogenous bases of nucleotides face each other and form Hydrogen
bonds with their complimentary bases. A is complimentary to T while G is complimentary to C.
A form 2 bonds with T while G forms 3 bonds with C. Hence a G≡C bond is thermally more stable than an
A=T bond.
Gene Organization
Protein Family: The similar and non-identical genes are called as the gene family. A closely related
homologous protein encoded by a gene family is called as the protein family.
Duplicate Gene: A gene is said to be duplicate gene when it arise from duplication of a previous
gene and are identical to that gene. A gene is said to be tandomly repeated gene when gene repeat
one after another in a head to tail fashion.
Repeated DNA Sequence: The DNA which has less than 105 copies is called moderately repeated
DNA sequence and the DNA which has more than 105 copies is called highly repeated DNA
sequence.
Solitary Gene: The protein coding sequence may be solitary or may be representing by a gene
family. The protein coding sequence represent only once in the genome called as solitary gene. In
multicellular organism 25-30% protein coding genes are present once in genome. For example the
chicken lysozyme enzymes which constitute a transcriptional unit containing 4 exons and 3 introns.
It is found in bacterial cell wall, in chicken as white protein and in human tears. The activity of
lysozyme helps to keep the surface of egg and eyes sterile.
C-Value Paradox: The extensive variation in nuclear genome size among eukaryotic species is
known as the C-value and the failure of c-value to its phylogenetic complexity is called as the C-value
paradox.
Morphology of Chromosome
Shape and Size: Chromosome shape and size is varies from species to species. Chromosome in
plants has a larger size than in animals. Monocotyledons has large size chromosome than the
dicotyledons. The largest chromosome is called as lamphbrush chromosome.
Type: Based upon the centromere chromosome divided into following categories
Genes inside the cell follow several layers of organisation to enable the long DNA to be compacted into
the chromosome fibers.
These helices form the long chromatin fiber which, with series of turns and loops, forms the third level
of spatial DNA organisation.
Finally, these chromatin fibers are compactly packed inside the chromosome. In the Chromosome, the
chromatin fibers are wrapped around a protein scaffold.
The chromosome is stained with specific dye under this technique. There are following type of banding
techniques are there:
C-Banding: first treated with moderated strong alkali follow by heating and then stained with
Giensa and acriding orange dye.
In order to duplicate and segregate correctly the chromosome must contain following three parts:
Centromere:
It is an ovoid, non-stainable structure made up of three zones outer zone, middle zone and inner zone.
Telomere:
It prevents the deoxyribonucleases from degrading the end of linear DNA molecule.
In eukaryotic DNA replication origin were first identified by their function in yeast transformation
studies. If the yeast cell lack the particular gene require for amino acid synthesis they can transformed
with cloned plasmid containing missing DNA. The plasmid contains the Autonomously Replicating
Sequences (ASR). This ASR can act as the origin point of replication.
Genome Organisation
Genome organization refers to the sequential, not the structural organization of the genome. Besides
the coding exons, the non-coding DNA in Eukaryotes may fall in the following classes:
Introns
They are DNA sequences inserted between the exons and found in the ORF. They are spliced after the
first level of transcription. Most introns are junk inserted within genes.
Pseudogenes
'Dead', non-functional copies of genes present elsewhere in the genome, but no longer of any use.
Transposons
These are the Jumping genes, which splice themselves in and out of the genome (in DNA form)
randomly, by the action of transposase.
Retrotransposons
They transcribed into an mRNA, which encodes an RT enzyme, which then copies the mRNA back to DNA
and incorporates it into the genome.
Infect in humans only 1.5% of the entire genome length corresponds to coding DNA. This 1.5% codes for
about 27,000 genes which in turn code for proteins that are responsible for all the cellular processes.
Noncoding DNA
In genetics, noncoding DNA describes components of an organism's DNA sequences that do not encode
for protein sequences. In many eukaryotes, a large percentage of an organism's total genome size is
noncoding DNA, although the amount of noncoding DNA and the proportion of coding versus noncoding
DNA vary greatly between species.
Much of this DNA has no known biological function and at one time was sometimes referred to as "Junk
DNA". However, many types of noncoding DNA sequences do have known biological functions, including
the transcriptional and translational regulation of protein-coding sequences.
The amount of total genomic DNA varies widely between organisms, and the proportion of coding and
noncoding DNA within these genomes varies greatly as well. More than 98% of the human genome does
not encode protein sequences, including most sequences within introns and most intergenic DNA.
The extensive variation in nuclear genome size among eukaryotic species is known as the C-value
enigma or C-value paradox. About 80% of the nucleotide bases in the human genome may be
transcribed but transcription does not necessarily imply function.
Many noncoding DNA sequences have very important biological functions. Comparative genomics
reveals that some regions of noncoding DNA are highly conserved, sometimes on time-scales
For example, in the genomes of humans and mice, which diverged from a common ancestor 65–75
million years ago, protein-coding DNA sequences account for only about 20% of conserved DNA, with
the remaining majority of conserved DNA represented in noncoding regions. Linkage mapping often
identifies chromosomal regions associated with a disease with no evidence of functional coding variants
of genes within the region, suggesting that disease-causing genetic variants lie in the noncoding DNA.
Some noncoding DNA sequences are genetic "switches" that do not encode proteins, but do regulate
when and where genes are expressed. According to a comparative study of over 300 prokaryotic and
over 30 eukaryotic genomes, eukaryotes appear to require a minimum amount of non-coding DNA. This
minimum amount can be predicted using a growth model for regulatory genetic networks, implying that
it is required for regulatory purposes. In humans the predicted minimum is about 5% of the total
genome.
Some noncoding DNA sequences determine the expression levels of various genes. Other sequences of
noncoding DNA determine where transcription factors attach.
Some specific sequences of noncoding DNA may be features essential to chromosome structure,
centromere function and homolog recognition in meiosis.
Global dimension of genomes was demonstrated particularly with the Fractal globule proof. Then,
another potential way is a possible function of noncoding DNA at the global balance scale of the whole
genome: considering the global genome DNA information as a whole and analysing it at the codon
population’s level, could provide new ways to understand a possible global level function of noncoding
DNA.
Transposition
The movement of mobile DNA from one place to another place in the genome is called transposition and
the mobile DNA molecule is called as the transposons. There are three main kinds of transposition are
there:
Replicative Transposition
They operate on the copy and paste mechanism. Examples are: Tn3 in bacteria.
Retroposition
They operate on the copy and paste mechanism but through the RNA intermediates. Examples are:
copia elements in drosophila, TYL in Yeast.
Retrotransposons
Retrotransposons (also called transposons via RNA intermediates) are genetic elements that can amplify
themselves in a genome and are ubiquitous components of the DNA of many eukaryotic organisms.
They are a subclass of transposon. In mammals, almost half the genome (45% to 48%) comprises
transposons or remnants of transposons. Around 42% of the human genome is made up of
retrotransposons while DNA transposons account for about 2-3%.
Biological Activity
The retrotransposons' replicative mode of transposition through RNA intermediate increases the copy
numbers of elements rapidly and thereby can increase genome size. Like DNA transposable elements
(class II transposons), retrotransposons can induce mutations by inserting near or within genes.
Furthermore, retrotransposons-induced mutations are relatively stable, because the sequence at the
insertion site is retained as they transpose via the replication mechanism.
Retrotransposons copy themselves to RNA and then back to DNA that may integrate back to the
genome. The second step of forming DNA may be carried out by a reverse transcriptase which the
retrotransposons encodes. Transposition and survival of retrotransposons within the host genome are
possibly regulated both by retrotransposons- and host-encoded factors, to avoid deleterious effects on
host and retrotransposons as well, in a relationship that has existed for many millions of years between
retrotransposons and their plant hosts. The understanding of how retrotransposons and their hosts'
genomes have co-evolved mechanisms to regulate transposition, insertion specificities, and mutational
outcomes in order to optimize each other's survival is still in its infancy.
Most retrotransposons are very old and through accumulated mutations, are no longer able to
retrotranspose.
Types of Retrotransposons
Retrotransposons, also known as class I transposable elements, consist of two sub-types, the long
terminal repeat (LTR) and the non-LTR retrotransposons.
LTR Retrotransposons
LTR retrotransposons have direct LTRs that range from ~100 bp to over 5 kb in size.
Non-LTR Retrotransposons
It consists of two sub-types, long interspersed nuclear elements (LINEs) and short interspersed nuclear
elements (SINEs). They can also be found in high copy numbers up to 250,000 in the plant species.
LINEs
Long interspersed repetitive elements or long interspersed nuclear elements are a group of genetic
elements that are found in large numbers in eukaryotic genomes. They are transcribed (or are the
SINEs
Short interspersed repetitive elements or Short interspersed nuclear elements are short DNA sequences
(<500 bases) that represent reverse-transcribed RNA molecules originally transcribed by RNA
polymerase III into tRNA, rRNA, and other small nuclear RNAs. SINEs do not encode a functional reverse
transcriptase protein and rely on other mobile elements for transposition. The most common SINEs in
primates are called Alu sequences. Alu elements are 280 base pairs long, do not contain any coding
sequences, and can be recognized by the restriction enzyme AluI (hence the name). With about
1,500,000 copies, SINEs make up about 11% of the human genome.
Transposons in Prokaryotes
1. IS Elements
General structure of IS element:
Mechanism of Transposition:
The enzyme which catalyzed the transposition of IS elements sequence is known as the transposase.
Enzyme transposase make the blunt end cut in donor DNA and staggered cut in target DNA.
Transposase ligase inserts the donor DNA segment into the target DNA.
The enzyme DNA polymerase extends the three primate cuts ends and enzyme ligase join them.
Tng elements:
The IS elements are inserted in same orientation and has resistant to antibiotic chloramphenicol.
Tn5 elements:
The IS elements are inserted in inverted orientation and has resistant to antibiotic kanamycin.
Tn10 elements:
The IS elements are inserted in inverted orientation and has resistant to antibiotic tetracycline.
3. Tn3 Elements
The Tn3 element do not have the IS element sequence instead they have 38-48 base pair of long simple
inverted repeated sequence at their terminal ends. They has three genes TnpA, TnpR and b/a.
Transposons in Eukaryotes
First class of these mutants is reversible at higher frequency whereas the second class of mutations does
not reverse back unless they occur in the presence of first class.
Meclentok called the agents responsible for first class mutation the Activators (Ac) and the agents
responsible for second class mutation the Dissociation elements (Ds).
2. Retrotransposons:
All eukaryotes studied from yeast to humans contain the retrotransposons. These are the mobile
elements transposes by a RNA intermediate. These are divided into two categories:
Viral Retrotransposons:
The viral retrotransposons are abundant in yeast (e.g. Ty element) and in drosophilla (e.g. copia
element). The viral retrotransposons is about 4% of human DNA.
A function of these two LTR’s is as promoters and directs the host cell RNA polymerase to increase the
transcription.
The resulting RNA genome lack the complete long terminal repeat sequences. However when a virus
inject into a cell the reverse transcription of the RNA genome by the virus and encoding reverse
transcription yields a double standard DNA.
Then the enzyme integrate encoded by the virus inserts these double standard DNA into the host cell.
Immunoassays are based on the specific Ab-Ag reaction. All the Immunoassay depends upon the
measurement of fractional binding site occupancy of the antibody by analyte. In competitive
Immunoassay labeled and unlabeled analyte are simultaneously exposed to the Ab. In noncompetitive
Immunoassay the labeled Ab detects the bound analyte. Maximum sensitivity will be reached by
decreasing the amount of antibody in competitive assays and increasing the antibody concentration in
noncompetitive assays.
Radioimmunoassay
ELISA exploits an enzymatic reaction for detecting the immune reaction. It has similar principal to RIA
but depends on an enzyme rather than a radioactive label. An enzyme conjugate with an Ab reacts with
Different variants of ELISA have been developed allowing qualitative detection or quantitative
measurement of either Ag or Ab. Each type of ELISA can be used qualitatively to detect the presence of
Ab or Ag. Alternatively a standard curve based on the known concentration of Ab or Ag is prepared from
which the unknown concentrations of a sample can be determined.
Indirect ELISA
Ab can be detected or quantitatively determined with an indirect ELISA. Serum or some other sample
containing primary Ab (Ab1) is added to an Ag-coated micro-titer well and allowed to react with Ag
attached to the well. After any free Ab is washed away the presence of Ab bound to the Ag is detected
by adding an enzyme conjugated secondary anti isotope (Ab2) which binds to the primary Ab. Any free
Ab2 then is washed away and a substrate for the enzyme is added. The amount of colored reaction
product that forms is measured by specialized by spectrophometric plate readers which can be measure
the absorbance of all of the wells of a 96-well plate in less than a few minutes. Indirect ELISA is a method
of choice to detect the presence of serum Ab against HIV.
Ag can be detected or measured by the sandwich ELISA. In this technique the Ab (rather than an
antigen) is immobilized on a micro-titer well. A sample containing antigen is added and allowed to react
with the immobilized Ab. After the well is washed a second enzyme linked Ab specific for a different
epitope on the Ag is added and allowed to react with the bound Ag. Any free second Ab is then washed
away and a substrate for the enzyme is added. The amount of colored reaction product that forms is
measured.
Immunofluorescence
Ab could be labeled with molecules that have the property of fluorescence. Fluorescent molecules
absorbed light of one wavelength and emit light of another wavelength. If Ab molecule is label with a
fluorescent dye immune complexes containing these fluorescently labeled antibodies can be detected
by the color light emission when excited by light of appropriate wavelength. Ab molecules bound to Ag
in cells or tissue can be similarly visualized. Most commonly used dyes are fluorescein and rhodamine.
Labrum
The labrum is a small sclerite articulate concealing the mandibles
It serves to hold food in place during chewing by the mandibles and thus can simply be described
as an upper lip
Embryonically, it is formed by the fusion of appendage remnants on either side of the head, thus
is a single structure with a paired developmental origin, similar to the labium
Its inner surface also bears two rows of sensory setae on each side
Mandible
Chewing insects have two mandibles, one on each side of the head
The mandibles are positioned between the labrum and maxillae
They are typically the largest mouthparts of chewing insects, being used to masticate (cut, tear,
crush, chew) food items
In carnivorous chewing insects, the mandibles can be modified to be more knife-like, where-as in
herbivorous chewing insects, they are more typically broad and flat on their opposing faces (e.g.,
caterpillars)
In male stag beetles, the mandibles are modified to such an extent that they do not serve any
feeding function, but are instead used to defend mating sites from other males
In ants, the mandibles also serve a defensive function (particularly in soldier castes). In bull ants,
the mandibles are elongate and toothed, used as hunting (and defensive) appendages
Each mandible bears three tooth like denticles and a small smooth serrated molar area
Maxilla
Situated beneath the mandibles, paired maxillae manipulate food during mastication
Maxillae can have hairs and “teeth” along their inner margins
At the outer margin, the galea is a cupped or scoop-like structure, which sits over the outer edge
of the labium
They also have palps, which are used to sense the characteristics of potential foods
Each maxillae consists of stripe which bears three processes:
A lacinia which is sclerotized with a pair of sharp denticles and a blunt lacinula
A galea which act as hood for the lacinia
A maxillary palp which is sensory in nature
Labium
The labium is a quadrupedal structure, although it is formed from two fused secondary maxillae
Hypopharynx
The hypopharynx is a somewhat globular structure, arising from the base of the labium. It assists
swallowing.
Feeding on nactor
Maxillae form the main proboscis not the labrum
Mandibles and labium are reduced
Maxillae palp is rudimentary
Labium form labial palp
Galea are much elongated and coiled each make half tube which become complete when they
are locked together
Rise in basal part uncoils the proboscis
These are the mouthparts of the fluid feeding insects so they are modified in various ways to
form tube like structure to draw fluid or inject saliva
Found in insects like aphids and leaf hoppers, bugs and mosquitoes
Mandibles and maxillae are modified into a proboscis, sheathed within a modified labium, which
is capable of piercing tissues and sucking out the liquids
Paired mandibles and maxillae are present, together forming the stylet, which is used to pierce
an animal's skin
Palp and cardo attached to the head region with paired muscles
Paired mandibles and maxillae are present, but much reduced and non-functional
The labium forms a proboscis which is used to channel liquid food to the oesophagous
The labellum's surface is covered by minute food channels, formed by the interlocking elongate
hypopharynx and epipharynx, which form a tube leading to the oesophagous
In Honey Bee
Consist of long tongue like glossae of labium ending in spoon shaped labellum
Food canal is formed by glossa, galea and labial palp fitted together
Needle like labrum is fused with epipharynx and forms covering of proboscis
Proboscis bears two small labellae used as feelers and help to select the appropriate part of its
victim
Maxillae are doubly grooved on their inner faces acts as food channel for flow of blood and
other salivary glands
A pest is any organism that interferes with your site management goals or any organism that may cause
damage to a resource (garden, house, wildlife habitat) that you are trying to protect. There are following
type of pests:
Key or Major Pest: They occur perennially and remaining above the economic level and always
demand control.
Occasional Pests: They are under biological control and when disrupted then only need control
measure.
Potential Pests: They cause no damage in prevailing conditions but insecticide use can increase
their population.
Integrated Pest Management (IPM) is a strategy which encourages the reduction of pesticide use by
employing a variety of pest control options in harmonious combination to contain or manage pests
below their economic injury levels. The IPMS require due to hazards and high cost of insecticides. These
options include:
Cultural control
Physical Control
Mechanical Control
Biological control
Chemical control
Adoption of IPMS
The plants possess some degree of natural tolerance against pests attacks. The cost of expenditure
should not be cross the productivity gain. For practical purposes, IPM programs can develop through
three stages.
STAGE 1
Soil Preparation: Growers give their plants a head start on pest problems by choosing the proper
site, testing the soil, rotating crops, creating raised beds where necessary, and providing sufficient
organic matter.
Forecasting: Weather data is consulted to predict if and when pest outbreaks will occur. Treatments
can then be properly timed, preventing crop damage and saving sprays.
STAGE 2
As for stage 1, plus
Pest Trapping: Traps that are attractive to insects are used so that growers can pinpoint when
thepest has arrived and decide whether control is justified.
Monitoring: Growers inspect representative areas of the fields regularly to determine whether pests
are approaching a damaging level.
Thresholds: Before treating, growers wait until pest populations reach a scientifically determined
level that could cause economic damage. Until that threshold is reached, the cost of yield and quality
loss will be less than the cost for control.
STAGE 3
As for stage 2, plus
Cultural Controls: The pest's environment is then disrupted by turning under crop residues,
sterilizing greenhouse tools, and harvesting early.
Biological Controls: It is necessary for growers to conserve the many beneficial natural enemies
already at work. They import and use additional biological where effective.
Chemical Controls: Growers select the most effective and appropriate pesticide and properly
calibrate sprayers. They then verify that weather conditions will permit good coverage without undue
drift.
Recordkeeping: Records of pest traps, weather and treatment are kept for use in pest management
decisions.
Advantages of IPMS
The disadvantage of IPM is that it is more complex than control by chemicals alone. It requires a greater
understanding of the interactions between pests and beneficial, as well as the effects of chemicals.
Practices and routines need to be modified and new information absorbed by the practitioners.
The following are some areas that are likely to be important:
Regular monitoring is necessary to identify pest outbreaks and their location within a crop. It
may take time to develop suitable procedures and routines.
"Soft" controls for some pests are available but not for others. If unavailable, spot spraying with
broad spectrum products may be more appropriate than widespread spraying.
Some damage from pests may need to be tolerated. Some pests may be required to support a
useful population of the natural enemy.
Good timing is necessary when introducing natural enemies - not too early, not too late. For
example: You may need to target moth flights, or detect early signs of a pest etc.
Need to get natural enemies established quickly. If introducing mass reared BCA's, introduce
appropriate numbers to facilitate quick establishment.
Deciding when to or not to spray can be difficult. If soft options are available for the pest in
question this is not such an issue.
Providing a suitable environment. Very hot dry conditions are not conducive to some BCA's.
Adjustments may need to make to favor BCA's, e.g. shade, windbreaks, overhead watering.
Having an expectation that one cannot spray chemicals at all is incorrect and may result in
failure of the IPM system. BCA's usually recover from occasional sprays of moderately toxic
products and can remain at useful levels.
Cultural Control
It includes followings:
Habitat Modification
Crop rotation
Mix cropping
Contour study
Strip cropping,
Soil manuring and fertilization
Growing resistant verities of plants
It includes followings:
Hand picking
Shaking and beating branches
Trapping
Flood and drain
Physical Control
Cooling
Heating
Radiant energy
Biological Control
Natural enemies include predators, parasites and diseases of pests. Farming practices that protect
natural enemies are used. Predators, such as ladybird beetles, are released in greenhouses. Bacteria-
based pesticides are used on vegetable and other crops. Avoiding pesticides protects natural enemies in
home gardens.
1. Inoculative Release: One or two releases early in pest infestation to control pest gradually. E.g.
predatory mites in strawberries, Trichogramma in outdoor crops, lacewing in field and greenhouse crops
2. Regular Or Dribble Release Method: Regular small release during likely problem periods, used
like preventative fungicides. e.g. P. persimilis is nursery crops, Encarsia in green house crops.
3. Inundative Releases: Repeated high rate releases during periods of pressure for quick knock down.
E.g. Cryptolaemus beetles, Trichogramma in green house crops, P. persimilis for dosing hot spots of
TSM.
4. Combination Of Above Methods: e.g. initial high release rate for quick knock down followed by
regular targeted small releases.
Chemical Control
It includes followings:
Pesticides
Chemosterilants
Antifedants
Attractants
Repellents
Physiological Selectivity: In this process the insecticide interfere the physiological process. For
example use of the juvelline on developmental stages to prevent the metamorphosis and use of cutical
synthesis inhibitors.
Behavioral Selectivity: Knowledge of habits and behavior of pest are helpful in reducing the amount
of insecticides.
Time Dose Formulation Selectivity: The pest has week stages in life cycle so insecticides should
apply in week stages.
Ecology Vs IPMS
IPMS are a balanced management aim for proper selection of various control methods, pathological
conditions of pests and host and thorough knowledge of ecology and ecological protections.
Main influence on ecosystem resulted in conversion of ecosystem to agro system i.e. “unit composed of
total complex of organism in crop area together with overall environmental conditions.
Several factors are responsible for disturbance in agro system and thereby increased influence of pest
population like:
Monoculture
Continuous irrigation
1. Phylum Porifera:
Amphiblastula
It is formed by a process inversion from stomoblastula
Examples: Scypha
Parenchymula
Solid, oval or flattened larval stage
Ephyra Larva
For example: Aurelia
5. Phylum Annelida:
Trochophore Larva
For example: Nereis
Glochidium Larva
For example: Unio
7. Phylum Arthropoda:
Protopod
This structure believes to form due to immature hatching of the egg
In these larvae poorly developed thoracic and cephalic appendages are formed
Abdomen is unsegmented
Polypod
There are additional legs in addition to 3 pairs of thoracic legs present on the abdomen called
prolegs
Campodeiform: In these larvae body is elongated and fusiform, thoracic legs are long and abdomen
carries a pair of terminal processes on hind end. These larvae are fast running and predatory in habit.
Scarbaeiform: These larvae are C-Shaped and sub cylindrical thoracic legs are short and stumpy.
Caudal processes are absent and larvae are less active.
They are divided into three categories on the basis of degree of development of head
Eucephalous: Has well developed head and cephalic appendages. E.g. Red wasp
Acephalous: Head and appendages are wanting. In this buccopheryngeal apparatus for feeding is
present. E.g. Housefly
8. Phylum Echinodermata:
Class I: Asteriodea
Bipinnaria Larva: The larva has three projections on each side of body bordered by ciliary bands. It
swims in clockwise rotation with its anterior end foremost.
Brachiolaria Larva: Bipinnaria larva transform into the brachiolaria larva. Three short additional arms
are added to preoral lobe known as branchiolar arms.
Class V: Crioidea
Doliolaria Larva: After gastrulation and formation of coelomic sac and gut embryo becomes doliolaria
Larva.
Crustaceans are large number of arthropods, comprising almost 52,000 described species. The majority
of them are aquatic living in either marine or fresh water environments, but a few groups have adapted
to life on land, such as terrestrial crabs, terrestrial hermit crabs and wood lice. Crustaceans show both
direct and indirect development. In most crustacea, development is accompanied by little or more
metamorphosis and the various stages of development are known as larvae. Several larval forms are
met within Crustacea and special terms are applied to each one of them.
1. Nauplius Larva
It is the simplest, commonest and earliest larval form in crustacea
Nauplius is a microscopic, oval or pear shaped with an unsegmented body having a broad anterior
head region, an intermediate trunk-region and a posterior bilobed anal region
It has 3 pair of unjointed appendages bearing swimming seate. The first pair is uniramous and
become the antennules of the adult. Second is antennaryand third is mandibular called antennae
and mandibles respectively in the adult
The mouths open anteriorly between the bases of antennary and mandibular feet. While the anus
lies at the extremity of the caudal region. The alimentary canal is straight and made of foregut,
midgut and hindgut. However mouth and alimentary canal are lacking in nauplius of Cirripedia
Dorsal shield of the head grows back to form carapace. In addition to the 3-origional appendages of
nauplius, it also develops the rudiments of 4 pairs of appendages, which become the maxillulae the
maxillae and the first 2 pairs of maxillipedes of the adult
3. Cypris Larva
In some Cirripedia (Sacculina, Lepas), the nauplius passes into the cypris stage, in which the body
and the appendages are enclosed within bivalved carapace with an adductor muscle to close it
Its modified antennules have of cement glands at their bases. It undergoes a remarkable series of
metamorphoses to become the sessile adult form
4. Protozoaea Larva
The metanauplius larva is succeeded by the protozoaea stage with 7 pairs of appendages and the
beginning of segmentation
The carapace become enlarged and covers the dorsal surface anteriorly. The 7 pairs of appendages
present in the metanauplius (up to 2nd maxillipede) become well-developed and capable of
movements
The rudiments of the remaining posterior six thoracic segments are also marked off, but the
abdomen is still unsegmented and without limbs
5. Zoaea Larva
Zoaea is the second important larvae of the Crustacea, the first being the nauplius. Protozoaea stage
is succeeded by the zoaea stage
The zoaea is characterized with a distinct cephalothorax and abdomen, 8 pair of appendages and
buds of 6 more, and resembles the adult Cyclops
The paired lateral and stalked compound eyes become well-formed and remaining 6 pair of thoracic
appendages appears in the form of bud
The long abdomen is distinctly made of 6 segments, and terminates in a caudal furca, but still lacking
in appendages
6. Metazoaea Larva
The older zoaea or metazoaea has well-formed third maxillipedes, which are biramous and
swimming organs in Anomura, but uniramous and non-swimming in Brachyura
7. Calyptosis Larva
In Euphausiacea, one of the larval stages is termed calyptopsis .It is similar in all respects to a typical
zoaea except that the paired 'eyes are not stalked but sessile
Head is unsegmented, bearing median and paired eyes and all the 5 pairs of cephalic appendages
The thorax is made of segments, free from the carapace, and bearing anterior 5 pairs of biramous
swimming appendages
9. Alima Larva
The so-called alima larva of Squilla which hatch out from the egg directly, is a modified zoaea
It is apeagic larva, having a glass-like transparency and occurring in large numbers in the plankton
All the head appendages are present. But only is 6-segmented, having 4 or 5 pairs of pleopods
The alima larva differs from the zoaea larva in the armature of the telson and a very large raptorial
second maxillipedes
The abdomen is also well formed, straight and bears biramous pleopods
It corresponds to the megalopa stage of Bracchyura with a large symmetrical abdomen and a full
complement of adult appendages
All the thoracic appendages are biramous. Even the 5 pairs of posterior thoracic legs are biramous
with flagellar exopodites which take up the locomotory function uptill now fulfilled chiefly by the
antennae
The abdomen develops similar to that of the adult form, with 5 pairs of biramous pleopods and a
pair of uropods and telson
The mysis larva metamorphosis in to the adult prawn by the loss of the exopodites on the thoracic
legs
It is a remarkable for its large size, extremely flattened and leaf- like delicate form and glassy
transparency.
A large oval carapace covers the head and the first two thoracic segments.
Only anterior 6 pairs of thoracic appendages are present in the newly hatched larva. The first
thoracic appendages or maxillipedes are rudimentary (palinurus) or absent (Scyllarus) and the
second are uniramous, succeed by 4 pairs of very long and biramous legs with notatory exopodites
Last two pairs of thoracic appendages are usually absent.
Abdomen, though indistinctly segmented is very small and limbless. Phyllosoma undergoes several
moults before reaching the adult form.
Larvae represent one of the classic problems of evolutionary biology and may explain how new body
plans originate. It has often been suggested that many entirely unique body plans first originated as
retained larvae of ancestral organisms.
Pseudometamerism
Pseudometamerism occurs in cestodes in which every segment is independent of the other and contains
complete set of organs that have no connection with organs in other segments. During growth new
segments are added in front, in the neck region and hence the posterior-most body segment is the
oldest one and the anterior segments are younger.
True Metamerism
In true metamerism, there is a serial repetition of homologous organs, like nephridia, nerves, muscles,
reproductive organs, appendages etc. in each segment but these organs function in coordination with
the others. All segments are integrated into a single functional unit. In true metamerism new segments
are added in front of the last segment called pygidium. Hence posterior segments are younger as
compared to the anterior ones. Coelom is divided by intersegmental septa into compartments, each of
which can be regulated independently of the others.
Truly segmented animals typically have an anterior acron and posterior pygidium and various
intermediate segments called metameres or somites. In higher invertebrates, such as arthropods,
metamerism provided an opportunity for specialization of segments into head, thorax and abdomen and
serially repeated organs could be specialised resulting in rapid evolution.
Evolution of Metamerism
It is believed that during the course of evolution metamerism evolved three times independently for
different purposes as mentioned below.
Pseudometamerism occurs in cestodes such as tapeworms. There are two theories to explain its origin.
Fission Theory
Proposed by Perrier 1882, this theory postulates that pseudometamerism evolved in flat worms by
strobilation of body as happens in strobila of scyphozoa. Strobilation is aimed to increase the rate of
reproduction. Proglottids of helminths are serially arranged segments but in reverse order and they
increase reproductive capacity many times.
Pseudometamerism Theory
Hymen (1951) proposed that pseudometamerism evolved in turbellarians and nemertians first by serial
repetition of organs, particularly the reproductive organs for increasing fecundity. Later, these organs
were separated by cross-partitioning of body producing metamerism. Turbellarians are not segmented
but some have serially repeated organs. Nemertians are intermediate between turbellarians and
annelids and possess a specialized coelom called rhynchocoel in which organs are serially repeated but
not separated by septa. Ancestors of metameric animals were perhaps similar to nemertians and
archiannelids such as Polygordius, which are also not clearly segmented animals.
True metamerism evolved in animals twice independently, once in Annelida and Arthropoda and again
in chordates.
Metamerism in Annelida
R.B. Clark (1964) proposed the LOCOMOTION THEORY to explain the origin of metamerism in annelids.
According to this theory metamerism evolved in annelids as an adaptation to peristaltic locomotion and
for burrowing. Annelids possess what is called peristaltic locomotion which involves shortening and
lengthening of body by circular and longitudinal muscles. As the coelom is filled with coelomic fluid
peristaltic locomotion will not be possible unless the coelom is divided by septa, so that high pressure
produced by contraction of muscles can be confined to a particular region and it does not affect the
whole body. By having metamerism annelids not only can save energy by keeping high pressure areas in
selected regions but also can control and regulate locomotory movements in different directions.
For burrowing in sand and mud annelids require a hard skeleton which they do not possess. Hence they
produce what is known as hydraulic skeleton with the help of coelomic fluid and intersegmental septa.
Arthropods inherited metamerism from annelids in which body organs and appendages were serially
repeated in each segment. Arthropods used this condition to specialised body organs and reduce their
number (Williston’s rule of serial homology). Therefore, arthropods specialised segmented body into
tagma, such as cephalothorax and abdomen in crustaceans or into head, thorax and abdomen in insects.
Appendages were modified to produce antennae, mouth parts, walking and swimming appendages,
Metamerism in Chordates
Clark’s Locomotion Theory
R.B. Clark’s (1964) theory postulates that metamerism evolved independently in chordates also for
locomotion which was carried out by lateral undulation of body in primitive aquatic vertebrates.
Metamerism allowed myotomes or muscle bundles and nerves to be arranged segmentally for better
co-ordination of undulatory movement of body.
Cyclomerism Theory
This theory, proposed by Sedgwick in 1884, says that metamerism in chordates evolved for better
arrangement of organs in coelom. True coelom or enterocoel evolves by outpouching of coelenteron in
three places to produce protocoel, mesoscoel and metacoele, which further partitioned later to produce
somites. This provided septa and compartments in coelom in which organs could be arranged in a better
way.
Metamorphosis is a biological process by which an animal physically develops after birth or hatching,
involving a conspicuous and relatively abrupt change in the animal's body structure through cell growth
and differentiation. Metamorphosis means "change of form." It’s the way insects grow and mature.
Their lives are divided into separate stages for resting, growing and reproducing.
Humans grow gradually. You began life as a baby and grow a little at a time until you’re an adult. While
you’re growing, the basic plan of your body doesn’t change. You have the same body your whole life.
Insects grow in stages. The cycle of stages is called metamorphosis. For many insects, the stages are so
different from one another that you might not recognize them as the same animal.
Type of Metamorphosis
Complete Metamorphosis
Complete metamorphosis is the way butterflies, bees, flies, beetles and many other insects develop.
Complete metamorphosis has four stages: egg, larva, pupa and adult.
Egg- Every insect begins life as an egg. The egg is the embryo stage.
Larva- The larva hatches from the egg. The larva is the eating and growing stage. Some insects
don’t eat at all after this stage. Larvae don’t look like adults. Caterpillars, grubs and maggots are
larvae that grow up to be butterflies, beetles and flies as adults. A larva’s exoskeleton can’t stretch
or grow, so the larva sheds its skin, or molts, several times as it grows.
Pupa- When a larva has finished growing, it forms a pupa (plural: pupae). The pupa is the insect’s
transforming stage. Outside, the pupa looks as if it’s resting. But inside, the entire body is
rearranging. New organs, muscles and body parts develop.
Adult- When it has finished changing, the pupa molts one last time, emerging as an adult. The adult
is the reproductive stage. The adult has all the identifiable insect features: three body sections, six
legs, two antennae and usually wings.
Incomplete Metamorphosis
Some insects, such as grasshoppers, dragonflies and cockroaches, develop by incomplete
metamorphosis. Incomplete metamorphosis has three stages: egg, nymph and adult. There are two
types of incomplete metamorphosis based upon food and habitat. First type is known as
paurametabolous in which parents and young ones share same kind of food and thus has same habitat.
Second type is hemimetabolous in which food and habitat of young ones are different from adults.
Nymph- The nymph is the eating and growing stage. Nymphs often look like smaller versions of
adults, without wings. The nymph’s exoskeleton can’t grow or stretch, so the nymph needs to shed
its skin, or molt, in order to grow. The periods between molts are called instars. Each instar looks a
little more like the adult form. Wing buds form and grow on the nymph’s back.
Adult- The adult is the reproductive stage. The nymph emerges from its final molt as an adult. In
species that have wings, the wings don’t fully appear until this stage. Adults mate, females lay eggs,
and the cycle begins again.
Hormonal control
Insect growth and metamorphosis are controlled by hormones synthesized by endocrine glands near the
front of the body.
Neurosecretory cells of an insect's brain secrete a hormone, the prothoracicotropic hormone that
activates prothoracic glands, which secrete a second hormone, usually Ecdysone (a steroid), that induces
metamorphosis. Ecdysone activates the epithelial layer below the cuticle to produce a fluid called
moulting fluid. This fluid has a capacity to dissolved endocuticle which is the major layer of cuticle.
Exocuticle is thus separated out and dissolved endocuticle is reused in the formation of new layer of
cuticle.
Moreover, the corpora allata, a retrocerebral organ produces the juvenile hormone, whose effect is to
prevent the development of adult characteristics while allowing ecdysis.
Juvenile hormone neutralized the effect of Ecdysone and cause delay in moulting.
Therefore, the insect is subject to a series of molting, controlled by Ecdysone, until the production of
juvenile hormone ceases and metamorphosis occurs.
Microbiology is a field of biology which deals with microorganisms (which cannot be seen with naked
eyes) and their effect on other living beings especially human beings.
The primary or basic working place of microbiologists is microbiology laboratory. In these laboratories all
types of experiments related to microbiology are done. For example staining, culturing, identification
and lots of other research work. These experiments are also related to biochemistry, biotechnology and
some other branches of modern biology.
There are several rules followed in any microbiology laboratory like any other science laboratory. One of
the most important rule followed in microbiology laboratory is to maintain extremely clean, sterile (free
of any living organism) condition inside the microbiology laboratory. In simple words, microbiology
laboratory must be free of any unwanted living being.
There are a wide variety of tools used in microbiology laboratory. But the most common and most
important tool used in microbiology laboratory is microscope. Microbiology laboratory is totally
incomplete without microscope as it is the most frequently used instrument in any microbiology
laboratory. There are many more instruments used in microbiology laboratory.
Following are the main rules followed in any general microbiology laboratory:
Sterile condition must be maintained inside the laboratory. All the equipment’s used in the lab
should be sterilized prior to the use for any experiment.
Microbiologist also has to strictly maintain sterile condition. Before entering into the lab
microbiologists have to take bath, change clothes and pass through a sterile chamber. Prior to any
experiment must clean hands with soap and then sterilize with alcohol (wipe hands with cotton
soaked in alcohol).
Flame resistant or retardant lab coat or apron is must inside the lab for everyone.
It is suggested not to work with open wound or cut inside the lab as it may result in infection.
Books or other items must not keep in working place.
Place provided for the experiment must be cleaned with provided disinfectant prior to the
experiment.
Contaminated cultures must be discarded and burnt. And contaminated equipment’s must be
properly sterilized for further use.
Eating or smoking is not allowed inside the lab.
Talking during performing an experiment must be prohibited.
Essential equipment’s in microbiology laboratory include centrifuge, laminar air flow, autoclave,
incubator, hot air oven, colony counter, Spectrophotometer, pH meter, balances, stirrers, shakers, glass
equipment’s.
Sterilization- This is most repeatedly used technique in microbiology lab to obtain lab materials
free from any living organism.
Staining- It is done to increase contrast to highlight any living cell in the microscopic image.
Observation under microscope- The microscopy is used to observe unicellular organisms or cell
clusters for their identification, characterization and to know their various properties.
Incubation- It is done to maintain microbial culture especially bacterial culture at a particular
temperature for a specific time length to obtain growth.
Centrifugation- It is used in microbial lab mainly to disperse cells and also to obtain a specific
substance (for example- plasmid, specific protein etc).
There are many more basic techniques done in microbiology laboratory during various experiments.
These techniques include electrophoresis, blotting, hybridization, fermentation etc.
Media Preparation
Microorganisms need nutrients, a source of energy and certain environmental conditions in order to
grow and reproduce. In the environment, microbes have adapted to the habitats most suitable for their
needs, in the laboratory, however, these requirements must be met by a culture medium. This is
basically an aqueous solution to which all the necessary nutrients have been added. Depending on the
type and combination of nutrients, different categories of media can be made.
Categories
Complex media are rich in nutrients, they contain water soluble extracts of plant or animal tissue
(e.g., enzymatically digested animal proteins such as peptone and tryptone). Usually a sugar, often
glucose is added to serve as the main carbon and energy source. The combination of extracts and
sugar creates a medium which is rich in minerals and organic nutrients, but since the exact
composition is unknown, the medium is called complex.
Defined media are media composed of pure ingredients in carefully measured concentrations
dissolved in double distilled water i.e., the exact chemical composition of the medium is known.
Typically, they contain a simple sugar as the carbon and energy source, an inorganic nitrogen source,
various mineral salts and if necessary growth factors (purified amino acids, vitamins, purines and
pyrimidines).
Selective/differential media are media based on either of the two categories above supplemented
with growth-promoting or growth-inhibiting additives. The additives may be species- or organism-
selective (e.g., a specific substrate, or an inhibitor such as cyclohexamide (artidione) which inhibits
all eucaryotic growth and is typically used to prevent fungal growth in mixed cultures).
The mixture of necessary nutrients can be used as a liquid medium, or a solidifying agent can be added.
"Agar agar" is a natural polysaccharide produced by marine algae and is the most commonly used
solidifying agent added to media (end concentration usually 1.5 % w/v). If hydrolysis of the agar is
suspected, a silica gel is used as a replacement solidifying agent.
Tryptone=5.0 g
Yeast extract=5.0 g
Glucose=1.0 g
Agar=15.0 g
Distill Water=1.0 Liter
pH=7.0
Beep extract=3.0 g
Peptone=5.0 g
Nacl=5.0 g
Agar=15.0 g
Distill Water=1.0 Liter
pH=7.0
Sucrose=20.0 g
K2HPo4=1.0 g
MgSo4=0.5 g
Nacl=0.5 g
Na2MoO4=0.001 g
FeSo4=0.10 g
Agar=15.0 g
Distill Water=1.0 Liter
Sterilization Techniques
Sterilization is the complete destruction or removal of all living organisms from the object being
sterilized. The development of methods of sterilization was mainly a consequence of the controversy
over spontaneous generation culminating in the work of Pasteur.
Experiments designed to prove or to disprove spontaneous generation depended upon two general
principles:
The complete sterilization of a suitable growth medium so that no living organisms exist at the
start of the experiment,
The design of the vessel of a type that it is impossible for microbes to enter from outside
This was necessary following the realization of the existence of microbes floating around in the air If
these two principles arc strictly followed and conditions are otherwise suitable for multiplication of
microbes, any growth occurring must be the result of spontaneous generation.
Attaining, and
Maintaining sterility
The usual method depended upon heat treatment. However, it was soon realized that microorganisms
vary widely in their resistance to heating. Bacteria require temperature and some also produce heat
stable spores. Thus boiling at normal pressure was insufficient to kill these spores and, therefore,
autoclave was designed to increase the pressure, and, thereby, the temperature sterilization.
Sealing of the flask was not proper as oxygen, known to be essential for many forms of life, could no
longer enter the vessel. It was necessary, therefore, to include some kind of filter to prevent the entry of
microbes but not of air. This led to the development of the cotton wool plug that was soon adopted
universally by microbiologists. By the end of the 19th century most of the methods currently used for
sterilization had been developed.
Before inoculation with the desired microorganisms, microbiological media and all materials coming
into contact with it must be sterile. During any subsequent handling of the bacterial cultures, unwanted
or contaminant organisms must be excluded employing aseptic techniques.
Sterilization implies the complete destruction of all microorganisms including spores; this is
accomplished by the use of heat, chemicals, radiation, and filtration.
Heat: Denatures and coagulates vital proteins. There are various forms of heat sterilization.
Red Heat: Inoculating wires or loops are sterilized by holding them in a Bunsen flame until they
are red hot.
Moist Heat: Bacteria are more readily destroyed by moist heat (steam) than dry heat. Usually
used for the sterilization of culture media, aqueous solutions and the destruction of discarded
cultures. Air must first be removed in order to achieve the 121 °C necessary for successful
sterilization. This is accomplished by the use of an autoclave (the technical version of a pressure
cooker), which follows automatic cycles of heating under pressure for the required time.
Dry Heat: Usually employed for materials which could either be corroded by steam or must
remain dry before use. These include metal instruments, glass petri dishes, flasks and pipettes
and cotton wool. In practice, dry heat sterilization requires longer time intervals and higher
temperatures than steam sterilization, e.g. Steam sterilization 121°C for 15mins or dry heat
sterilization 160°C for 120 minutes.
Chemical: Usually employed for delicate equipment such as optical instruments and electrical
devices which would be damaged by heat. Due to the toxicity of the chemicals used, this is not the
most popular form of sterilization. Chemicals employed include: gaseous ethylene oxide, which
alkylates amino, sulfhydryl, carboxyl and hydroxyl groups of microbial cell compounds;
formaldehyde, used as a fumigant; and hydrogen peroxide vapor used in aseptic packaging.
Bacteria 0.2 µm
Membrane filtration is usually employed for heat-sensitive substances, e.g. vitamin solutions;
the filters are heat-sterilized before use.
Nucleation Track (Nuclepore) Filters: These filters consist of very thin polycarbonate films
which have been treated with nuclear radiation and then etched with a chemical to create very
uniform vertical holes. They are employed for the same material as membrane filters but have
the disadvantage that they are more easily clogged.
Inoculation
The introduction of bacteria in culture media is called as inoculation. Bacteria may be introduced to the
media (inoculated) by various means. Usually the bacteria e.g. from a drop in a heat-sterilized loop are
spread on the surface of (ready set) agar.
Serial Dilutions
Serial dilution involves repeatedly mixing known amounts of source culture with (sterilized) liquid. 1 ml
added to 9 ml gives a 10-fold dilution; 1 ml added to 99ml gives a 100-fold dilution.
When fixed amounts of this dilution series are mixed with an appropriate agar and incubated, then
different numbers of colonies will be obtained.
By working back from an easily counted plate and using the appropriate dilution factor, the number of
micro-organisms in the original source culture can be calculated.
Procedure:
Lie out and label the tubes and (empty) Petri dishes as shown in the diagram below.
Each member of the team can take it in turns to perform the repeated sections below, and prompt
others as required.
Flame and loosen the lids of tubes 0 and 1.
Using a sterile pipette transfer 1 ml of liquid from tube 0 to plate 0, and transfer 1 ml of liquid from
the source culture (tube 0) to tube 1.
Metabolic Activity
Another indirect way to estimate bacterial numbers is to use the metabolic activity of the population.
The amount of a metabolic product is measured and assumed to be proportional to the number of
bacteria present. Examples of metabolic products include CO2 and organic acids. Oxygen uptake can
Refrigeration
It can be used for short-term storage. Cultures streaked on agar slants or stab cultures may be viable
over several months when stored at 4°C. Plates have to be sealed to prevent their tendency to dry out.
To preserve cultures for longer periods of time, two methods are commonly employed:
Deep Freezing
A pure culture of bacteria is suspended in a liquid and quick-frozen (often with liquid nitrogen) at
temperatures between -50°C and-95°C. Sensitive microorganisms require the presence of glycerol (end
concentration 15-20 %), which acts as an "antifreeze", or extra protein (skimmed milk powder) to
protect them. Cultures can be thawed and used up to several years later.
Lyophilization
A suspension of bacteria is quickly frozen and the water removed by means of a high vacuum. The
microbes survive in this powder like residue for several years and can be revived at any time by
rehydration of the culture in a nutrient medium. Bacterial strains ordered from strain collections are
usually delivered in this form.
There are several methods that can be used for counting the cells and spores of bacteria and other
microbes. These include direct count, plate count, and most probable number (MPN) determinations.
Direct count
The density of cells, spores etc. of microorganisms can be found out by counting their number in a unit
volume. The simplest technique is to use a special counting chamber, of a type that used in a
haemocytometer for counting the blood cells. The counting chamber is simply a ruled slide with a
supported glass cover that holds a definite volume of fluid.
The nature of the growth medium and the incubation conditions determine which bacteria can grow
and thus be counted. Viable counting measures only those cells that are capable of growth on the given
medium under the set of conditions used for incubation. Sometimes cells are viable but non culturable.
Cell viability measurements assess healthy cells in a sample. This can be accomplished either by directly
counting the number of healthy cells or by measuring an indicator for healthy cells in cell populations
(e.g., in a microplate assay). Whether the cells are actively dividing or quiescent is not distinguished. An
increase in cell viability indicates cell growth, while a decrease in viability can be interpreted as the
result of either toxic effects of compounds/agents or suboptimal culture conditions.
In contrast to cell viability analysis, cell proliferation assessment is defined as the measurement of
actively dividing cells in a sample. It can be expressed either as the actual number or proportion of
proliferating cells in cell culture, tissues, or as relative values in assays for cell populations. Quiescent
nongrowing healthy cells are not detected by cell proliferation assays.
The most prominent parameter for analyzing cell proliferation is the measurement of DNA synthesis as a
specific marker for replication. In these assays, labeled DNA precursors are added to cells (or animals)
and their incorporation into genomic DNA during the S phase of the cell cycle (replication) is quantified
following incubation and sample preparation. The amount of labeled precursors incorporated into the
DNA is quantified either by measuring the total amount of labeled DNA in a population, or by
microscopically detecting the labeled nuclei. Incorporation of the labeled precursor into DNA is directly
proportional to the rate of cell division occurring in the sample.
Cell proliferation can also be measured using indirect parameters. With these techniques, molecules
that regulate the cell cycle are quantified either by measuring their activity (e.g., cyclin-dependent
kinase assays) or by direct quantification (e.g., Western blots, ELISA, or immunohistochemistry).
Most viability assays are based on one of two characteristic parameters: metabolic activity or cell
membrane integrity of healthy cells. Usually the metabolic activity is measured in cell populations via
incubation with a tetrazolium salt that is cleaved into a colored formazan product by metabolically
active cells. The ATP status of cells can also be analyzed and gives an indication for cellular energy
capacity and thus viability.
Cell Harvesting
It is done by centrifugation and by scraping the cell after deep freezing. The cultural medium is poured
off and vessel is placed into crushed ice. Then BSS solution is added and a sheet is scraped off by the
policeman (an instrument). Then this material is transferred to the centrifugation process, a little more
BSS can be added.
To study processes taking place in animal cells, e.g., metabolic regulations, cell physiology.
To enable cultivation of viruses for their study and production of vaccines
Easy growing of cells in vitro has led to a spurt in the activity to apply cell culture technology to
synthesize or produce a variety of biomolecules at an industrial scale, which otherwise would have
been difficult.
Cultured cells are used for reconstruction of damaged tissues or replacement of non-functional cells
or tissues.
Cultivation of patient's own cells in vitro in order to generate enough cells and/ or to genetically
manipulate the cells to replace a missing function, to overcome problem of immune rejection and of
scares tissue source. Parkinson's disease may one day be cured using cultured cells.
Antigen-specific killer T cells grown in vitro can be used in immune suppressed patients. May be
useful in as immune-suppression therapy, possibly in prevention of HIV cases turning into blown up
cases by using specific killer T cells.
Scanning of anti-cancerous drugs in tissue culture, as a clear correlation between in vitro and in vivo
activities of chemotherapeutic agents was demonstrated in 1950.
Use of tissue culture avoids many ethical objections raised against animal experiments and also
allows experiments on human tissues which otherwise could not be done in vivo.
Tissue culture requires strict aseptic conditions and skilled persons and techniques.
Cost of animal tissue culture and labour, often may be high and prohibitive.
The growth and metabolic regulatory mechanism that exist under in vivo conditions are absent in
culture condition.
Cell lines may not reflect the actual conditions in vivo and results may not be reproducible in the
living animals
Tissue culture may not always be possible.
Multicellular endoparasites, body composed of outer cell layer called syncytium enclosing
reproductive cells
Locomotion by cilia
No tissue, organ or cavities are present
Life cycle composed of alternation of sexual and asexual generation
Endoparasite in sea star, annelids and in other marine invertebrates
They have been placed between protozoa and Porifera
Shows affinities with protozoa, Coelenterata and with Platyhelminthes
Ctenophora
Ctenophora means comb bearer refers to the locomotory comb like plates on the body
Transparent gelatinous unsegmented body without nematocysts but has lasso cells
Skeletal, excretory, circulatory and respiratory system are absent
Development include metamorphosis with cydippid larva stage
Asexual reproduction absent
Economic importance: consume large number of oyster larvae at spawning time
Shows affinities with Coelenterata and with Platyhelminthes
They have been placed in Coelenterata
Rhynchocoela
Rhynchocoela means proboscis cavity. They have remarkable proboscis aperture used in capturing
pray
Also called ribbon worms due to long dorsoventraly flattened body
Excretory system consist of localized tubules ending in flame cells
Hermaphrodite, asexual reproduction by fragmentation
Nervous system include four lobed brain
Shows affinities with vertebrates, chordates and with Platyhelminthes
Entoprocta refers to the location of anus along with mouth within the crown of distal tentacles
Individual consist of calyx, stalk and stolon
Digestive tube is U-shaped and ciliated
Retractor muscles are present
Excretion occur by protonephridia ending in flame bulb, single nephridiophore near mouth
Development includes a free swimming ciliated larva which attaches to grow into adult
Shows affinities with Ectoprocta and with rotifera
Acanthocephala
Nematomorpha
Rotifera
Rotifera means wheel bearers refers to the rapid movement of cilia in a regular sequence on one or
more disc like lobes of head producing the appearance of a rotating wheel
Fresh water animal rarely marine
Body unsegmented divisible into three parts head, trunk and tail
Head modified into retractile ciliated corona for locomotion and food collection
Tail is mobile containing the pedal glands
Gastrotricha
Gastrotricha means stomach hairs refers to the ventral ciliation of the gastrotriches
Cuticle forms the scales, spines and bristles
Freshwater forms are with only parthenogenic females while marine forms are hermaphrodite
Eggs are large
Excretion is by protonephridia tubes with flame cells
Shows affinities with Rotifera and with Nematoda
Kinorhyncha
Kinorhyncha means movement snout refers to the withdrawal of head proboscis into neck
Body consist 13-14 rings which are strongly cuticulized
Head is retractile bear circlet of hook around a terminal mouth on a short proboscis
Body surface is devoid cilia but provided with spines and bristles
Excretion is by protonephridia tubes with flame bulb
Development include larval stages and metamorphosis
Shows affinities with Arthropoda, Nematoda and with Platyhelminthes
Bryozoa
Bryozoa means moss animal refers to the superficial resemblance to a colony of moss
Colonies found attached with stones, shell and other objects in shallow water
Zooids of colony connect together by an organic substance and shows polymorphism
Each zooid secrete a protective cup shaped exoskeleton Calles zoecium
The anterior end of zooid form a horseshoe shaped structure called lopophore bearing a series of
slender, ciliated, hollow tentacles
Development take place in a special brood chamber and include free swimming ciliated larva which
settle down and metamorphoses into the adult zooid from which colony is form by budding
Branchiopoda
Branchiopoda means arm foot refer to the large lopophore consisting a pair of coiledarm bearing
ciliated tentacles
Unsegmented body enclosed in a bivalve shell
Beneath the shell valve are present two mantle lobes enclosing a large mantle cavity
Alimentary canal is V-shaped
Shows affinities with Mollusca, Annelida and Bryozoa
Phoronida
Chaetognatha
Chaetognatha means bristle jaw refer to the presence of hook like grasping spines surrounding the
mouth
Body is arrow shaped divisible into three parts head, trunk and tail
Head bear bristle like jaw
Trunk and tail bearing fins supported by rays
Presence of a hood like fold of body wall which can be drawn over the head
Shows affinities with Annelida, Branchiopoda and Aschelminthes
Priapulida
Priapulida means phallus refers to the large retractile proboscis which has a resemblance to the
male organ
Sipunculoida
Echiuroida
Pogonophora or Branchiata
Pogonophora means bread refers to their long ciliated tentacles hence common name bread worms
Body is divisible into protostome, mesosome and trunk
Protostome bear several large hollow tentacles fringed with minute pinnule
Trunk with a pair of raised belts bears chitinous adhesive organs
Mouth, anus and digestive canal totally lacking
Fertilization occur in tube
Swimming is the major form of movement exhibited by sperm and by many protozoans. Although cilia
and flagella are the same, they were given different names before their structures were studied.
Typically, cells possess one or two long flagella, whereas ciliated cells have many short cilia. All cilia and
flagella are similar in their organization.
Structure
It contains a central bundle of microtubules, called the axoneme, in which nine outer doublet
microtubules surround a central pair of singlet microtubules with a “9 + 2” arrangement of
microtubules.
Each doublet microtubule consists of A and B tubules, or subfibers: the A tubule is a complete
microtubule with 13 protofilaments, while the B tubule contains 10 protofilaments. The bundle of
microtubules comprising the axoneme is surrounded by the plasma membrane.
At its point of attachment to the cell, the axoneme connects with the basal body. Basal bodies are
cylindrical structures which contain nine triplet microtubules. Each triplet contains one complete 13-
protofilament microtubule, the A tubule, fused to the incomplete B tubule, which in turn is fused to the
incomplete C tubule.
The A and B tubules of basal bodies continue into the axonemal shaft, whereas the C tubule terminates
within the transition zone between the basal body and the shaft. The two central tubules in a flagellum
or a cilium also end in the transition zone, above the basal body.
Within the axoneme, the two central singlet and nine outer doublet microtubules are continuous for the
entire length of the structure. There is an inner and an outer row of dynein arms which is permanently
attached to the A tubule of each doublet microtubule. These dyneins reach out to the B tubule of the
neighboring doublet.
The axoneme is held together by three sets of protein cross-links. The central pair of singlet
microtubules is connected by periodic bridges, like rungs on a ladder, and is surrounded by a fibrous
structure termed the inner sheath. A second set of linkers, composed of the protein nexin, joins
adjacent outer doublet microtubules. Nexin is proposed to be part of a dynein regulatory complex.
Radial spokes, which radiate from the central singlets to each A tubule of the outer doublets, form the
third linkage system.
Ciliary and Flagellar Beating Are Produced by Controlled Sliding of Outer Doublet Microtubules. In cilia
and flagella, the filaments are the doublet microtubules, all of which are arranged with their (+) end at
the outer tip of the axoneme. Each of the outer 9 doublet microtubules extends a pair of dynein arms
(an "inner" and an "outer" arm) to the adjacent microtubule; these dynein arms are responsible for
flagellar beating, as the force produced by the arms causes the microtubule doublets to slide against
each other and the flagellum as a whole to bend. These dynein arms produce force through ATP
hydrolysis. The active sliding occurs all along the axoneme, so that the resulting bends can be
propagated without damping.
An individual cilium moves in a particular asymmetric manner called effective stroke, during which the
cilium is more or less straight and its effect on the fluid is maximized. During the recovery stroke its
effect on the surrounding fluid is minimized since the cilium has a more curved shape. The
microorganism propagates in the opposite direction to the effective stroke. The movement of the cilium
is always in a plane perpendicular to the surface during the effective stroke. The recovery stroke
movement may lie in the same plane, but also in a plane perpendicular to the effective-stroke-plane, so
that the movement of a cilium may be truly three-dimensional.
When the entire cilia’s move in same direction at a time than it is called as the metachronic movement
and when move in different direction than it is called as the synchronic movement. The collective
movement of the cilia seems to happen in a concerted fashion. Neighboring cilia in such a way that a
collective wave-like motion going over the micro-organisms surface. This wave may travel in the same
direction as the swimming direction of the micro-organism (but opposite to the effective stroke; this is
called an antiplectic metachronic wave, and happens for Paramecium) or in the opposite direction
(called symplectic metachronic wave behavior).
Flagella Vs Cilia
Though eukaryotic flagella and motile cilia are ultra-structurally identical, the beating pattern of the two
organelles can be different. In the case of flagella (e.g. the tail of a sperm) the motion is propeller-like. In
contrast, beating of motile cilia consists of coordinated back-and-forth cycling of many cilia on the cell
surface. Thus, flagella serve for the propulsion of single cells (e.g. swimming of protozoa and
Functions
2. For cells anchored in a tissue, like the epithelial cells lining our air passages, this moves liquid over the
surface of the cell.
Gray (1939) has described the pattern of parapodial activity during locomotion in Nereis. According to
his observation each parapodium behaves like a miniature paddle alternating beating backward
(effective stroke) and forwards (recovery stroke). The opposite parapodia of adjacent segment always
performed the same stroke at a time while the opposite parapodia of the same segment perform
reverse stroke at the same time. Movements of parapodia are controlled by the oblique and parapodial
muscles and the coelomic fluid which may be forced into or withdrawn from them.
During locomotion one or two arm serve as leading arm and all the tube feet extends in the same
direction in a coordinate manner.
Nanotechnology is defined as the study and use of structures between 1 nanometer and 100
nanometers in size. To give you an idea of how small that is, it would take eight hundred 100 nanometer
particles side by side to match the width of a human hair.
Nanotechnology holds some answers for how we might increase the capabilities of electronics devices
while we reduce their weight and power consumption.
Nanotechnology may hold the key to making space-flight more practical. Advancements in
nanomaterials make lightweight solar sails and a cable for the space elevator possible. By significantly
reducing the amount of rocket fuel required, these advances could lower the cost of reaching orbit and
traveling in space.
Nanotechnology is having an impact on several aspects of food science, from how food is grown to how
it is packaged. Companies are developing nanomaterials that will make a difference not only in the taste
of food, but also in food safety, and the health benefits that food delivers.
Nanotechnology can improve the performance of catalysts used to transform vapors escaping from cars
or industrial plants into harmless gasses. That's because catalysts made from nanoparticles have a
greater surface area to interact with the reacting chemicals than catalysts made from larger particles.
The larger surface area allows more chemicals to interact with the catalyst simultaneously, which makes
the catalyst more effective.
Nanotechnology is being used to develop solutions to three very different problems in water quality.
One challenge is the removal of industrial wastes, such as a cleaning solvent called TCE, from
groundwater. Nanoparticles can be used to convert the contaminating chemical through a chemical
reaction to make it harmless. Studies have shown that this method can be used successfully to reach
contaminates dispersed in underground ponds and at much lower cost than methods which require
pumping the water out of the ground for treatment.
Nanotechnology can enable sensors to detect very small amounts of chemical vapors. Various types of
detecting elements, such as carbon nanotubes, zinc oxide nanowires or palladium nanoparticles can be
used in nanotechnology-based sensors. Because of the small size of nanotubes, nanowires, or
nanoparticles, a few gas molecules are sufficient to change the electrical properties of the sensing
elements. This allows the detection of a very low concentration of chemical vapors.
Drug Delivery
One application of nanotechnology in medicine currently being developed involves employing
nanoparticles to deliver drugs, heat, light or other substances to specific types of cells (such as cancer
cells). Particles are engineered so that they are attracted to diseased cells, which allows direct treatment
of those cells. This technique reduces damage to healthy cells in the body and allows for earlier
detection of disease.
Therapy Techniques
Buckyballs may be used to trap free radicals generated during an allergic reaction and block the
inflammation that results from an allergic reaction.
Nanoparticles, when activated by x-rays that generate electrons that cause the destruction of cancer
cells to which they have attached themselves. This is intended to be used in place radiation therapy with
much less damage to healthy tissue. Nanobiotix has released preclinical results for this technique.
Aluminosilicate nanoparticles can more quickly reduce bleeding in trauma patients by absorbing water,
causing blood in a wound to clot quickly. Z-Medica is producing medical gauze that uses aluminosilicate
nanoparticles.
Nanoparticles may be used, when inhaled, to stimulate an immune response to fight respiratory
virsuses.
Iron oxide nanoparticles can used to improve MRI images of cancer tumors. The nanoparticle is coated
with a peptide that binds to a cancer tumor; once the nanoparticles are attached to the tumor the
magnetic property of the iron oxide enhances the images from the Magnetic Resonance Imagining scan.
Nanoparticles can attach to proteins or other molecules, allowing detection of disease indicators in a lab
sample at a very early stage. There are several efforts to develop nanoparticle disease detection systems
underway. One system being developed by Nanosphere, Inc. uses gold nanoparticles, Nanosphere has
clinical study results with their Verigene system involving its ability to detect four different nucleic acids,
while another system being developed by T2 Biosystems uses magnetic nanoparticles to identify
specimens, including proteins, nucleic acids, and other materials.
Anti-Microbial Techniques
One of the earliest nanomedicine applications was the use of nanocrystalline silver which is as an
antimicrobial agent for the treatment of wounds.
A nanoparticle cream has been shown to fight staph infections. The nanoparticles contain nitric oxide
gas, which is known to kill bacteria. Studies on mice have shown that using the nanoparticle cream to
release nitric oxide gas at the site of staph abscesses significantly reduced the infection.
Burn dressing that is coated with nanocapsules containing antibotics. If an infection starts the harmful
bacteria in the wound causes the nanocapsules to break open, releasing the antibotics. This allows much
quicker treatment of an infection and reduces the number of times a dressing has to be changed.
A welcome idea in the early study stages is the elimination of bacterial infections in a patient within
minutes, instead of delivering treatment with antibiotics over a period of weeks.
Nanorobots could actually be programmed to repair specific diseased cells, functioning in a similar way
to antibodies in our natural healing processes.
Hydra possesses very primitive type of nervous system. It includes bipolar and multipolar neurons or
nerve cells lying immediately above the muscle processes and forming an irregular and
discontinuous nerve net or nerve plexus
Neighboring nerve cells are not fused together but their processes or neuritis forms the synaptic
junction. Such a nerve net is called as the synaptic nerve net. Nerve cells are numerous around the
mouth and pedal disc but show no grouping in form of a nerve controlling center like brain or nerve
ring
Nerve net of hydra is different from higher animals because it is unipolarised so that nerve impulse
can pass in all directions. This type of transmission is called as the diffuse transmission. Thus nerve
net shows diffuse unipolarised transmission
The epidermis and gastrodermis layer of hydra consists of two different inter connected nerve net.
The ectodermal nerve net is more effective. Their processes are connected to the sensory cells
which act as receptor for external stimuli and to epithilio and endothilio muscle cells which act as
effector by contracting their muscle processes. Such a combination of epithilio and endothilio
muscle cells, sensory cells and nerve net is referred to as neuro muscular system. This is just the
beginning of the evolution of the nervous system
Nerve nets connect sensory photoreceptors and touch sensitive nerve cells that are found in the
body wall and tentacles of hydras. This nervous system allows hydra to respond to physical contact
They may then detect food and other chemicals in a rudimentary way. Although the nerve net
allows the animal to respond to its environment, it has trouble alerting the animal from where the
stimulus is coming
For this reason, simple animals with nerve nets, such as hydra, will typically respond in the same
way to contact with an object, regardless of where the contact occurs
It is of simple and primitive type. It has the form of nerve net consisting of nerve fibres and few ganglion
cells all confide to the body wall except the visceral nerve plexus situated in the gut wall. At certain
places the nervous tissue is concentrated to form a distinct nerve cord. The nervous system of starfish
can be divided into four units:
Although the echinoderms do not have many well-defined sensory inputs, they are sensitive to touch,
light, temperature, orientation, and the status of water around them. The tube feet, spines, and
pedicellariae found on sea stars are sensitive to touch, while eyespots on the ends of the rays are light-
sensitive. The tube feet, especially those at the tips of the rays, are also sensitive to chemicals and this
sensitivity is used in locating odor sources, such as food.
The eyespots each consist of a mass of ocelli, consisting of pigmented epithelial cells that respond to
light and narrow sensory cells lying between them. Each ocellus is covered by a thick, transparent,
Brain
It consist of four ganglion concentrated in the head protected by a cartilaginous skull. A pair of
cerebral or supra oesophagal ganglion are fused together into a rounded mass. They give a pair of
stout optic nerve which expands into large kidney shaped optic ganglion of the eyes.
A small olfactory ganglion lies on the dorsal side of each optic nerve.
A pair of cerebro-buccal connectives connects the cerebral ganglion to a pair of superior buccal
ganglion which are situated to the dorsal side of buccal mass and connected by circum-oesophagal
connectives to a pair of inferior buccal ganglion lying below the buccal mass.
A pair of stout circum-oesophagal connectives connects the cerebral ganglion to the rest of brain.
Suboesophageal ganglionic mass is partly divided into anterior lobe, the branchial ganglion, and a
posterior lobe, the pedal ganglion.
Stellate ganglion
Pallial nerves on either side run backward to the inner surface of mantle cavity where it divided into two
branches. Outer branch immediately terminate into a large roughly triangular stellate ganglion. Several
nerves are arise from its outer border innervate the mouth. Inner branch is connected to the stellate
ganglion by two commissures
Sympathetic system
A pair of sympathetic nerves arising from the inferior buccal ganglion and run posteriorly along with the
oesophagous to join gastric ganglion which sends nerve to the liver, stomach, caecum and intestine
Optic Nerves: They arise from cerebral ganglion and extend in the form of the optic ganglion which
give sevral retinal nerves.
Visceral Ganglion: it is situated behind the cerebral ganglion and surrounding the oesophagous.
Pallial Nerves: they are very long arise from the posterior side of visceral ganglion and form the
stellate ganglion.
Stellate Ganglion: they are present on both the side at the base of the latral fins.
Visceral Nerves: they arise from the end of visceral ganglion and run backward and divided into
sevral nerves.
Branchial Nerves: these are 8 in number arise anteriorly from the pedal ganglion mass and
innervates the mantle.
Tentacular Nerves: these are 2 in numbers and arise from the pedal ganglion mass and supplies
the tantacles.
1. Ganglia
Cerebral Ganglia
Buccal Ganglia
Pleuropedal Ganglia
Supra-intestinal Ganglion
Visceral Ganglia
2. Commissures
Cerebral Commissure
Buccal Commissure
Pedal Commissure
3. Connectives
Two cerebro-buccal Connectives
Two cerebro-pleural Connectives
Two cerebro-pedal Connectives
Pleuro-infra intestinal Connective
Infra-intestinal visceral Connective
Supra-intestinal visceral Connective
Supra-intestinal pleural Connective
Increasing Cephalization
Bilateral animals tend to be more active require sense organs and feeding structures
Cephalization: concentration of sensory organs & feeding structures at the head or forward-moving
portion of an animal
Enlargement of the anterior ganglia that receive this sensory input and control feeding gave rise to
the first brains
An anterior brain connected to a nerve cord is the basic design for all organisms with central
nervous systems
Worms
The simplest organisms to have a central nervous system
More complicated nervous system allows worms to exhibit more complex forms of behavior
Although there is a separate brain in worms, the brain is not the sole control of action.
Even with its brain removed, worms are able to perform many types of behaviors, including
locomotion, mating, burrowing, feeding, and even maze learning
Mollusks
Nervous system complexity correlates with habitat as well as phylogeny
Slow moving mollusks (e.g. clams) have little or no cephalization and simple sense organs
Nervous system is a chain of ganglia circling the body
Cephalopods most sophisticated invertebrate nervous systems
Octopus – large brain & large image forming eyes
Rapid conduction along giant axons
Insects
Increased complexity of the brain and nervous system
Giant fiber systems (also found in worms and jellyfish) allow rapid conduction of nerve impulses
connect parts of the brain to muscles in legs or wings
Brain divided into three specialized segments: Protocerebrum, deutocerebrum, tritocerebrum
The Brainstem
Present in all mammalian brains
Oldest part of brain
Evolved ~ 500 million yrs ago
Called “reptilian brain” because it resembles the entire brain of a reptile
Handles basic functions for survival: breathing, heart rate, etc.
Determines alertness & detects incoming info
Limbic System
Group of structures located between the brainstem and the cortex
Evolved between 200 & 300 million years ago
Called the “mammalian brain” because it is most highly developed in mammals
Centralized Architecture
Evolved from a loose network of nerve cells (as in the jellyfish) to a spinal column and complex brain
with large swellings at the hindbrain and forebrain
Increasingly hierarchical
Newer additions to the human brain are involved in control
The initiation of voluntary behavior, the ability to plan, engage in conscious thought, and use
language depend on neocortical structures
Plasticity
Increase in plasticity
The brain ability to modify itself as a result of experience
Makes memory and the learning of new perceptual and motor abilities possible
The delivery of newly synthesized proteins to their proper cellular destinations usually referred to as
protein targeting or protein sorting encompasses two very different kinds of processes.
The first general process involves targeting of a protein to the membrane of an intracellular
organelle and can occur either during or soon after synthesis of the protein by translation at the
ribosome.
Second general sorting process applies to proteins that initially are targeted to the ER membrane,
thereby entering the secretory pathway.
Targeting to the ER generally involves nascent proteins still in the process of being synthesized. Proteins
whose final destination is the Golgi, lysosome, or cell surface are transported along the secretory
pathway by small vesicles that bud from the membrane of one organelle and then fuse with the
membrane of the next organelle in the pathway the information to target a protein to a particular
organelle destination is encoded within the amino acid sequence of the protein itself, usually within
sequences of 20–50 amino acids, known generically as signal sequences, or uptake-targeting sequences.
Each organelle carries a set of receptor proteins that bind only to specific kinds of signal sequences, thus
assuring that the information encoded in a signal sequence governs the specificity of targeting. Once a
protein containing a signal sequence has interacted with the corresponding receptor, the protein chain
is transferred to some kind of translocation channel that allows the protein to pass through the
membrane bilayer.
Transport of membrane and soluble proteins from one membrane-bounded compartment to another is
mediated by transport vesicles that collect “cargo” proteins in buds arising from the membrane of one
compartment and then deliver these cargo proteins to the next compartment by fusing with the
membrane of that compartment.
Proteins destine to go into mitochondria, peroxisomes and nucleus are released from the free
cytoplasmic ribosome which has synthesis them are transported into appropriate organelle by a
different mechanism in each case. This is called as the post-translational transport.
Since secretory proteins are synthesized in association with the ER membrane but not with any other
cellular membrane, a signal-sequence recognition mechanism must target them there. The two key
components in this targeting are the signal-recognition particle (SRP) and its receptor located in the ER
membrane.
Probably the energy from GTP hydrolysis is used to release proteins lacking proper signal sequences
from the SRP and SRP receptor complex, thereby preventing their mistargeting to the ER membrane.
Subsequent transfer of the nascent chain and ribosome to a site on the ER membrane where
translocation can take place allows hydrolysis of the bound GTP. After dissociating, SRP and its receptor
release the bound GDP and recycle to the cytosol ready to initiate another round of interaction between
ribosomes synthesizing nascent secretory proteins with the ER membrane.
If the translocon were always open in the ER membrane, especially in the absence of attached
ribosomes and a translocating polypeptide, small molecules such as ATP and amino acids would be able
to diffuse freely through the central pore. To maintain the permeability barrier of the ER membrane, the
translocon is regulated so that it is open only when a ribosome–nascent chain complex is bound. Thus
the translocon is a gated channel analogous to the gated ion channels.
As the growing polypeptide chain enters the lumen of the ER, the signal sequence is cleaved by signal
peptidase, which is a transmembrane ER protein associated with the translocon.
After the signal sequence has been cleaved, the growing polypeptide moves through the translocon into
the ER lumen. The translocon remains open until translation is completed and the entire polypeptide
chain has moved into the ER lumen.
Membrane Proteins
There are many integral (transmembrane) proteins that are present in the plasma membrane. Each such
protein has a unique orientation with respect to the membrane’s phospholipid bilayer. Integral proteins
located in ER, Golgi, and lysosomal membranes and in the plasma membrane, which are synthesized on
the rough ER, remain embedded in the membrane as they move to their final destinations along the
same pathway followed by soluble secretory proteins.
The topology of a membrane protein refers to the number of times that its polypeptide chain spans the
membrane and the orientation of these membrane-spanning segments within the membrane. The key
elements of a protein that determine its topology are membrane-spanning segments themselves, which
usually contain 20–25 hydrophobic amino acids. Each such segment forms an alpha helix that spans the
membrane, with the hydrophobic amino acid residues anchored to the hydrophobic interior of the
phospholipid bilayer.
Topological classes I, II, and III comprise single-pass proteins, which have only one membrane spanning
alpha helical segment. Type I proteins have a cleaved N-terminal signal sequence and are anchored in
the membrane with their hydrophilic N-terminal region on the luminal face (also known as the
exoplasmic face) and their hydrophilic C-terminal region on the cytosolic face. Type II proteins do not
contain a cleavable signal sequence and are oriented with their hydrophilic N-terminal region on the
cytosolic face and their hydrophilic C-terminal region on the exoplasmic face (i.e., opposite to type I
proteins). Type III proteins have the same orientation as type I proteins, but do not contain a cleavable
signal sequence. These different topologies reflect distinct mechanisms used by the cell to establish the
membrane orientation of transmembrane segments.
The proteins forming topological class IV contain multiple membrane-spanning segments. A final type of
membrane protein lacks a hydrophobic membrane-spanning segment altogether; instead, these
proteins are linked to an amphipathic phospholipid anchor that is embedded in the membrane.
Formation of disulfide bonds in the ER: Disulphide bond do not form in the cytosol due to reducing
environment. Disulphide bond in ER facilitated by the protein disulphide isomerase (PDI). PDI also
cause the rearrangement of disulphide bond. Disulphide bond found only in secretory proteins.
Proper folding of polypeptide chains and assembly of multisubunit proteins in the ER: It is assisted
by the chaperone and other ER proteins like BiP. Chaperons are the class of proteins which bind to
incompletely folded protein in order to assist their folding or prevent them from aggregating.
Chaperons functions mainly by preventing formation of incorrect structure rather than by
promoting formation of correct structure. Thus chaperons prevent the nascent chain from
misfolding. Some other ER proteins like calnexin and calreticulin bind selectively to certain N-linked
oligosaccharides on growing nascent chain and prevent aggregation of segments and misfolding.
After synthesis in the cytosol, the soluble precursors of mitochondrial proteins (including hydrophobic
integral membrane proteins) interact directly with the mitochondrial membrane. The import receptors
subsequently transfer the precursor proteins to an import channel in the outer membrane.
This channel, composed mainly of the Tom40 protein, is known as the general import pore because all
known mitochondrial precursor proteins gain access to the interior compartments of the mitochondrion
through this channel. In the case of precursors destined for the mitochondrial matrix, transfer through
the outer membrane occurs simultaneously with transfer through an inner membrane channel
composed of the Tim23 and Tim17 proteins. (Tim stands for translocon of the inner membrane.)
Translocation into the matrix thus occurs at “contact sites” where the outer and inner membranes are in
close proximity.
Soon after the N-terminal matrix-targeting sequence of a protein enters the mitochondrial matrix; it is
removed by a protease that resides within the matrix. The emerging protein also is bound by matrix
Hsc70, a chaperone that is localized to the translocation channels in the inner mitochondrial membrane
by interacting with Tim44. This interaction stimulates ATP hydrolysis by matrix Hsc70, and together
these two proteins are thought to power translocation of proteins into the matrix.
Small membrane-bounded vesicles that transport proteins from one organelle to another are common
elements in the secretory and endocytic pathways. These vesicles bud from the membrane of a
particular “parent” (donor) organelle and fuse with the membrane of a particular “target” (destination)
organelle.
The budding of vesicles from their parent membrane is driven by the polymerization of soluble protein
complexes onto the membrane to form a proteinaceous vesicle coat Interactions between the cytosolic
portions of integral membrane proteins and the vesicle coat gather the appropriate cargo proteins into
the forming vesicle. Thus the coat not only adds curvature to the membrane to form a vesicle but also
acts as the filter to determine which proteins are admitted into the vesicle.
The integral membrane proteins in a budding vesicle include v-SNAREs, which are crucial to eventual
fusion of the vesicle with the correct target membrane. Shortly after formation of a vesicle is completed,
the coat is shed exposing its v-SNARE proteins. The specific joining of v-SNAREs in the vesicle membrane
with cognate t-SNAREs in the target membrane brings the membranes into close apposition, allowing
the two bilayers to fuse.
Forward (anterograde) transport is mediated by COPII vesicles, which are formed by polymerization
of soluble COPII coat protein complexes (blue) on the ER membrane
v-SNAREs (red) and other cargo proteins (green) in the ER membrane are incorporated into the
vesicle by interacting with coat proteins
Dissociation of the coat recycles free coat complexes and exposes v-SNARE proteins on the vesicle
surface
After the uncoated vesicle becomes tethered to the cis-Golgi membrane in a Rab-mediated process,
pairing between the exposed v-SNAREs and cognate t-SNAREs in the Golgi membrane allow vesicle
fusion, releasing the contents into the cis-Golgi compartment
Reverse (retrograde) transport, mediated by vesicles coated with COPI proteins (green), recycles the
membrane bilayer and certain proteins, such as v-SNAREs and missorted ER-resident proteins from
the cis- Golgi to the ER
All SNARE proteins are shown in red although each v-SNARE and t-SNARE is distinct proteins.
It is important that translated proteins are delivering to their specific cellular location. To accomplish
this protein is transferred through a series of membrane structure.
A principal membrane component is the Golgi apparatus. The Golgi apparatus is the sorting organelle of
the cell. Proteins from the rough endoplasmic reticulum are sent to the Golgi. As the protein move
through the Golgi apparatus they are modified and packed into the vesicles. Because the Golgi
apparatus receive the protein from the location and target them for deliver to a second location it is
sometimes considered as the post office of the cell.
And the key players in this process, the protein being transported and the enzyme that modified them.
Transported proteins are encapsulated in vesicles in ER. A group of these vesicles fuse and these fused
vesicles form the cis cisterna. As the protein move through the stack it is modified by the resident Golgi
enzyme at specific location in the apparatus. These modifications are the important because they
provide the signal that determines the final destination of protein so in that way the protein move into
Golgi.
The movement occurs in waves. First cis cisterna becomes the part of the median Golgi cisterna. Behind
it a new cis cisterna is formed by the fusion of the vesicle from ER. Meanwhile one of the median
cisterna migrates and become trans-cisterna. Collectively this process is known as the cis maturation
model.
Protein are so added on the in the Tran Golgi network protein with the same target sequence are
destine to the delivery to the same location. The Tran Golgi network then forms the vesicles. These
vesicles then migrate to their target location. These locations can be internal organelles such as
lysosome i.e. the digestive organelle. The vesicle can also be targeted to some membrane where
targeted protein released from the cell to deliver elsewhere in organism.
After formation of vesicles by budding from a donor membrane, the coats depolymerize into
their subunits, which are re-used to form additional transport vesicles
COPII vesicles mediate anterograde transport from the rough ER to the cis-Golgi/cis-Golgi
network
COPI vesicles mediate retrograde transport within the Golgi and from the cis-Golgi/cis-Golgi
network to the rough ER
The coat proteins surrounding secretory vesicles are not yet characterized; these vesicles carry
secreted proteins and plasma-membrane proteins from the trans-Golgi network to the cell
surface
Vesicles coated with clathrin (red) bud from the trans-Golgi network and from the plasma
membrane; after uncoating, these vesicles fuse with late endosomes
The coat on most clathrin vesicles contains additional proteins not indicated here. Note that
secretory proteins move from the cis- to trans-Golgi by cisternal progression, which is not
mediated by vesicles.
Peroxisomes are the small organelle with single membrane. They contain about 50 different enzymes
which are synthesized on the free cytoplasmic ribosomes. There are two peroxisomes targeting signals
PTS1: It is a C-terminal peptide. After transport into the peroxisomes signal is not cleaved so it is
the part of the mature protein.
A variety of cytoplasmic proteins called peroxisomes are needed for transport of targeted protein. It has
been demonstrated that a folded protein can be transported into the peroxisomes which might implies
a very large membrane pore but how this happen is still not known.
All protein fused in the nucleus are synthesized into the cytoplasm and actively involving into the
nucleus. This includes restriction proteins e.g. Laminas, histone, DNA RNA polymerase and HNRNP’s
particles, as well as protein that shuttle between the nucleus and cytoplasm e.g. HNRNP, AI, protein like
X141.
All protein actively imported through the nucleus pore complex contain a nuclear localizing signal. The
shutting protein contains both nuclear exporting signals and nuclear localizing signal. Some soluble
cytoplasmic components and ATP are required for nuclear import. Four cytoplasmic proteins are
requires for nuclear import of a protein containing a basic nuclear localizing signal have been purified
and characterized. These are impotin alpha, nuclear transfer factor II and Ran.
Importin alpha and beta form a heterodimeric nuclear import receptor that binds to a basic nuclear
localizing signal through the alpha subunit and the beta subunit interact with the nucleus pore complex
cytoplasmic filaments in the absence of ATP.
Trafficking mechanisms
A model for the import of the cytoplasmic cargo protein bearing nuclear localizing signal has been
proposed. By comparing the model for nuclear import with that of that of nuclear export we can see the
similarities between the two transport processes. Nature of both import and export depend upon the
asymmetric distribution of Ran GTP activating protein i.e. Ran GAP, which is restricted to the cytoplasm
and the Ran nucleotide exchange factor which is restricted to the nucleoplasm.
The model of nuclear export and import requires Ran GDP diphosphate i.e. transported from the
cytoplasm to nucleus, and Ran GTP i.e. transported from nucleus to cytoplasm and the other
components. Importin alpha and beta selectively transported into or out of the nucleus depending upon
their association with other proteins.
Ran GDP is the part of the cargo complex during export but not during import.
The receptor that directs the transport of a cargo protein through nucleus pore complex is a
monomer in case of export but a dimer in case of import.
Another difference between the two transported processes is that import of cargo protein with
nuclear localizing signal also requires NCF2. This soluble cytoplasmic factor interact in vitro with
Ran GDP importing beta and nucleus pore complex but its precise role is not known.
In some cases, proteins destined for the apical membrane are sorted into their own transport vesicles
that bud from the trans-Golgi network and then move to the apical region, whereas proteins destined
for the basolateral membrane are sorted into other vesicles that move to the basolateral region.
The different vesicle types can be distinguished by their protein constituents, including distinct Rab and
v-SNARE proteins, which apparently target them to the appropriate plasma-membrane domain.
In this mechanism, segregation of proteins destined for either the apical or basolateral membranes
occurs as cargo proteins are incorporated into particular types of vesicles budding from the trans-Golgi
network.
Such direct basolateral-apical sorting has been investigated in cultured Madin-Darby canine kidney
(MDCK) cells, a line of cultured polarized epithelial cells.
In MDCK cells infected with the influenza virus, progeny viruses bud only from the apical membrane,
whereas in cells infected with vesicular stomatitis virus (VSV), progeny viruses bud only from the
basolateral membrane.
This difference occurs because the HA glycoprotein of influenza virus is transported from the Golgi
complex exclusively to the apical membrane, and the VSV G protein is transported only to the
basolateral membrane.
Translation is the third stage of protein biosynthesis. In translation, messenger RNA (mRNA) produced
by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that
will later fold into an active protein. In Eukaryotes, translation occurs across the membrane of the
endoplasmic reticulum. The ribosome facilitates decoding by inducing the binding of tRNAs with
complementary anticodon sequences to that of the mRNA. The tRNAs carry specific amino acids that are
chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome in a
fashion reminiscent to that of a stock ticker and ticker tape.
In many instances, the entire ribosome/mRNA complex will bind to the outer membrane of the rough
endoplasmic reticulum and release the nascent protein polypeptide inside for later vesicle transport and
secretion outside of the cell. Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and
small nuclear RNA, do not undergo translation into proteins.
Translation proceeds in four phases: activation, initiation, elongation and termination. Amino acids are
brought to ribosomes and assembled into proteins.
In activation, the correct amino acid is covalently bonded to the correct transfer RNA (tRNA). The amino
acid is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. When the tRNA has an
amino acid linked to it, it is termed "charged". Initiation involves the small subunit of the ribosome
binding to 5' end of mRNA with the help of initiation factors (IF). Termination of the polypeptide
Mechanisms
The mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to
the ribosomes. The ribonucleotides are "read" by translational machinery in a sequence of nucleotide
triplets called codons. Each of those triplets codes for a specific amino acid.
The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a
multisubunit structure containing rRNA and proteins. It is the "factory" where amino acids are
assembled into proteins. tRNAs are small noncoding RNA chains (74-93 nucleotides) that transport
amino acids to the ribosome. tRNAs have a site for amino acid attachment, and a site called an
anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes for their
cargo amino acid.
Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific tRNAs and the amino
acids that their anticodons sequences call for. The product of this reaction is an aminoacyl-tRNA
molecule. This aminoacyl-tRNA travels inside the ribosome, where mRNA codons are matched through
complementary base pairing to specific tRNA anticodons. The amino acids that the tRNAs carry are then
used to assemble a protein. After the new amino acid is added to the chain, the energy provided by the
hydrolysis of a GTP bound to the translocase EF-G (in prokaryotes) and eEF-2 (in eukaryotes) moves the
ribosome down one codon towards the 3'end. The energy required for translation of proteins is
significant. For a protein containing n amino acids, the number of high-energy Phosphate bonds
required to translate it is 4n+1. The rate of translation varies; it is significantly higher in prokaryotic cells
(up to 17-21 amino acid residues per second) than in eukaryotic cells (up to 6-9 amino acid residues per
second).
Ribonucleic acid (RNA) is one of the three major macromolecules (along with DNA and proteins) that are
essential for all known forms of life.
Like DNA, RNA is made up of a long chain of components called nucleotides. Each nucleotide consists of
a nucleobase (sometimes called a nitrogenous base), a ribose sugar, and a phosphate group. The
sequence of nucleotides allows RNA to encode genetic information. For example, some viruses use RNA
instead of DNA as their genetic material, and all organisms use messenger RNA (mRNA) to carry the
genetic information that directs the synthesis of proteins.
Like proteins, some RNA molecules play an active role in cells by catalyzing biological reactions,
controlling gene expression, or sensing and communicating responses to cellular signals. One of these
active processes is protein synthesis, a universal function whereby mRNA molecules direct the assembly
of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the
ribosome, where ribosomal RNA (rRNA) links amino acids together to form proteins.
The chemical structure of RNA is very similar to that of DNA, with two differences--(a) RNA contains the
sugar ribose while DNA contains the slightly different sugar deoxyribose (a type of ribose that lacks one
oxygen atom), and (b) RNA has the nucleobase uracil while DNA contains thymine (uracil and thymine
have similar base-pairing properties).
Unlike DNA, most RNA molecules are single-stranded. Single-stranded RNA molecules adopt very
complex three-dimensional structures, since they are not restricted to the repetitive double-helical form
of double-stranded DNA. RNA is made within living cells by RNA polymerases, enzymes which act to
copy a DNA or RNA template into a new RNA strand through processes known as transcription or RNA
replication, respectively.
Structure of RNA
Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached
to the 1' position, generally adenine (A), cytosine (C), guanine (G) or uracil (U). Adenine and guanine are
purines, cytosine and uracil are pyrimidines. A phosphate group is attached to the 3' position of one
ribose and the 5' position of the next. The phosphate groups have a negative charge each at
physiological pH, making RNA a charged molecule (polyanion). The bases may form hydrogen bonds
between cytosine and guanine, between adenine and uracil and between guanine and uracil.
An important structural feature of RNA that distinguishes it from DNA is the presence of a hydroxyl
group at the 2' position of the ribose sugar. The presence of this functional group causes the helix to
adopt the A-form geometry rather than the B-form most commonly observed in DNA. This results in a
very deep and narrow major groove and a shallow and wide minor groove. A second consequence of the
presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule (that
RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil), but there are numerous
modified bases and sugars in mature RNAs. Pseudouridine (Ψ), in which the linkage between uracil and
ribose is changed from a C–N bond to a C–C bond, and ribothymidine (T), are found in various places
(most notably in the TΨC loop of tRNA). Another notable modified base is hypoxanthine, a deaminated
adenine base whose nucleoside is called inosine. Inosine plays a key role in the wobble hypothesis of the
genetic code.
The functional form of single stranded RNA molecules, just like proteins, frequently requires a specific
tertiary structure. The scaffold for this structure is provided by secondary structural elements which are
hydrogen bonds within the molecule. This leads to several recognizable "domains" of secondary
structure like hairpin loops, bulges and internal loops. Since RNA is charged, metal ions such as Mg2+ are
needed to stabilize many secondary and tertiary structures.
Types of RNA
Messenger RNA (mRNA) is the RNA that carries information from DNA to the ribosome, the sites of
protein synthesis (translation) in the cell. The coding sequence of the mRNA determines the amino acid
sequence in the protein that is produced. Many RNAs do not code for protein however (about 97% of
the transcriptional output is non-protein-coding in eukaryotes).
These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can
also derive from mRNA introns. The most prominent examples of non-coding RNAs are transfer RNA
(tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation. There are
also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are
able to catalyze chemical reactions such as cutting and ligating other RNA molecules, and the catalysis of
peptide bond formation in the ribosome; these are known as ribozymes.
In translation
Messenger RNA (mRNA): It carries information about a protein sequence to the ribosomes, the protein
synthesis factories in the cell. It is coded so that every three nucleotides (a codon) correspond to one
Transfer RNA (tRNA): It is a small RNA chain of about 80 nucleotides that transfers a specific amino acid
to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites
for amino acid attachment and an anticodon region for codon recognition that binds to a specific
sequence on the messenger RNA chain through hydrogen bonding.
Ribosomal RNA (rRNA): It is the catalytic component of the ribosomes. Eukaryotic ribosomes contain
four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized
in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein
combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein
synthesis. Several ribosomes may be attached to a single mRNA at any time. The rRNA is extremely
abundant and makes up 80% of the 10 mg/ml RNA found in a typical eukaryotic cytoplasm.
Transfer-messenger RNA (tmRNA): It is found in many bacteria and plastids. It tags proteins encoded by
mRNAs that lack stop codons for degradation and prevents the ribosome from stalling.
Regulatory RNAs
Several types of RNA can down regulate gene expression by being complementary to a part of an mRNA
or a gene's DNA. MicroRNAs (miRNA; 21-22 nt) are found in eukaryotes and act through RNA
interference (RNAi), where an effector complex of miRNA and enzymes can break down mRNA which
the miRNA is complementary to, block the mRNA from being translated, or accelerate its degradation.
While small interfering RNAs (siRNA; 20-25 nt) are often produced by breakdown of viral RNA, there are
also endogenous sources of siRNAs.
siRNAs act through RNA interference in a fashion similar to miRNAs. Some miRNAs and siRNAs can cause
genes they target to be methylated, thereby decreasing or increasing transcription of those genes.
Animals have Piwi-interacting RNAs (piRNA; 29-30 nt) which are active in germline cells and are thought
to be a defense against transposons and play a role in gametogenesis.
Many prokaryotes have CRISPR RNAs, a regulatory system similar to RNA interference. Antisense RNAs
are widespread; most down regulate a gene, but a few are activators of transcription. One way
antisense RNA can act is by binding to an mRNA, forming double-stranded RNA that is enzymatically
degraded. There are many long noncoding RNAs that regulate genes in eukaryotes, one such RNA is Xist
which coats one X chromosome in female mammals and inactivates it.
In RNA processing
Many RNAs are involved in modifying other RNAs. Introns are spliced out of pre-mRNA by spliceosomes,
which contain several small nuclear RNAs (snRNA), or the introns can be ribozymes that are spliced by
them. RNA can also be altered by having its nucleotides modified to other nucleotides than A, C, G and
U. In eukaryotes, modifications of RNA nucleotides are generally directed by small nucleolar RNAs
(snoRNA; 60-300 nt), found in the nucleolus and cajal bodies. The snoRNAs associate with enzymes and
guide them to a spot on RNA by base pairing to that RNA. These enzymes then perform the nucleotide
modification. The rRNAs and tRNAs are extensively modified, but snRNAs and mRNAs can also be the
target of base modification.
RNA genomes
Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA, and a
variety of proteins encoded by that genome. The viral genome is replicated by some of those proteins,
while other proteins protect the genome as the virus particle moves to a new host cell. Viroids are
another group of pathogens, but they consist only of RNA, do not encode any protein and are replicated
by a host plant cell's polymerase.
In reverse transcription
Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA;
these DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and
RNA from one another, and telomerase contains an RNA that is used as template for building the ends
of eukaryotic chromosomes.
Double-stranded RNA
Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all
cells. The dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-
stranded RNA such as viral RNA or siRNA can trigger RNA interference in eukaryotes, as well as
interferon response in vertebrates.
Transfer RNA
Transfer RNA (tRNA) is a small RNA molecule (usually about 74-95 nucleotides) that transfers a specific
active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during
translation. It has a 3' terminal site for amino acid attachment. This covalent linkage is catalyzed by an
aminoacyl tRNA synthetase. It also contains a three base region called the anticodon that can base pair
to the corresponding three base codon regions on mRNA. Each type of tRNA molecule can be attached
to only one type of amino acid, but because the genetic code contains multiple codons that specify the
same amino acid, tRNA molecules bearing different anticodons may also carry the same amino acid.
Anticodon
An anticodon is a unit made up of three nucleotides that correspond to the three bases of the codon on
the mRNA. Each tRNA contains specific anticodon triplet sequences that can base-pair to one or more
codons for an amino acid. For example, the codon for lysine is AAA; the anticodon of a lysine tRNA might
be UUU. Some anticodons can pair with more than one codon due to a phenomenon known as wobble
base pairing. Frequently, the first nucleotide of the anticodon is one of two not found on mRNA: inosine
and pseudouridine, which can hydrogen bond to more than one base in the corresponding codon
position. In the genetic code, it is common for a single amino acid to be specified by all four third-
position possibilities, or at least by both Pyrimidines and Purines; for example, the amino acid glycine is
coded for by the codon sequences GGU, GGC, GGA, and GGG.
To provide a one-to-one correspondence between tRNA molecules and codons that specify amino acids,
61 types of tRNA molecules would be required per cell. However, many cells contain fewer than 61
types of tRNAs because the wobble base is capable of binding to several, though not necessarily all, of
the codons that specify a particular amino acid. A minimum of 31 tRNA are required to translate,
unambiguously, all 61 sense codons of the standard genetic code.
The mRNA provides codon and dictates the sequence of the amino acids present in a polypeptide
chain. A sequence of three adjacent nucleotides in mRNA molecule, specifying either an amino acid
or a stop signal in protein synthesis is known as a "codon"
The rRNA s is structural and catalytic components of ribosomes which provide place or working
bench for protein synthesis
The tRNA carries amino acids to the site of protein synthesis. Specific tRNA carries specific amino
acid and having specific anti-codon, can translate specific codon of mRNA
Functions of DNA:
DNA contains the genetic instructions which are essential for the development and functioning of all
living organisms including some viruses
The main function of DNA is the long-term storage of genetic information
DNA is acts and functions as the blue print because it contains the instructions needed to construct
other components of cells like RNA and protein molecules
The DNA segments that carry this genetic information are called genes, but other DNA sequences
have structural purposes, or are involved in regulating the use of this genetic information. The
information carried by DNA is held in the of pieces of DNA called genes
Transmission of genetic information in genes is achieved via complementary base pairing. For
example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied
into a complementary RNA sequence through the attraction between the DNA and the correct RNA
nucleotides. Usually, this RNA copy is then used to make a matching protein
A process by which radioactive materials often incorporated into cell structures, are located by exposure
to a photographic emulsion forming a pattern on the film corresponding to the location of the
radioactive compounds within the cell. In this technique radioactive molecules make their location
known by exposing photographic films or emulsions.
When treated with a developing agent, the autoradiograms yield visible density images which can be
observed by the eye or measured by a densitometer for precise analysis. Radioisotope-labeled samples
can be detected on polyacrylamide or agarose gels, nylon or nitrocellulose membranes, or thin layer
chromatography plates. Autoradiography is widely used by scientists and researchers in the analysis of
chemical mixtures, recombinant DNA techniques applied to genetic marker mapping, genomic
sequencing, drug screening and plant fertilization studies.
Autoradiography Method
Living cells are briefly exposed to a ‘pulse’ of a specific radioactive compound
The tissue is left for a variable time
Samples are taken, fixed, and processed for light or electron microscopy
Sections are cut and overlaid with a thin film of photographic emulsion
Left in the dark for days or weeks (while the radioisotope decays) This exposure time depends on
the activity of the isotope, the temperature and the background radiation (this will produce with
time a contaminating increase in ‘background’ silver grains in the film)
The photographic emulsion is developed (as for conventional photography)
Counterstaining e.g. with toluidine blue, shows the histological details of the tissue. The staining
must be able to penetrate, but not have an adverse effect on the emulsion
Alternatively, pre-staining of the entire block of tissue can be done (e.g. with Osmium on plastic
sections coated with stripping film [or dipping emulsion] as in papers by McGeachie and Grounds)
before exposure to the photographic emulsion. This avoids the need for individually (post-) staining
each slide
It is not necessary to coverslip these slides
The position of the silver grains in the sample is observed by light or electron microscopy Note: the
grains are in a different plane of focus in the emulsion overlying the tissue section. Often oil with a
x100 objective is used for detailed observation with the light microscope.
These autoradiographs provide a permanent record
Full details on the batch of emulsion used, dates, exposure time and conditions should be kept for
each experiment
The process in which bacterial cells are grown in the presence of radiolabelled precursors of
macromolecule synthesis (DNA, protein, fatty acids and peptidoglycan). Blocks to radiolabel
incorporation can identify a mutant defect or the cellular target of an antimicrobial compound.
Incubate cells in serum free/phosphate free medium supplemented with 0.5% BSA for 2 hours in
order to deplete intracellular phosphate pools
Metabolically label with serum free media supplemented with 0.5% BSA and 0.5-1.0 Mci/mL 32P-
inorganic phosphate for four hours
Lyse nuclear fraction in lysis buffer A. After solubalization at 4 degrees for 15 minutes, spin samples
for 10 minutes to remove particulate/insoluble contaminants
Immunoprecipitate SF-1 from prepared soluble nuclear lysate in IP buffer. Preclear lysate with
protein a sepharose beads which have been blocked in 3% BSA for 30 minutes to remove
contaminants which non-specifically bind to the beads alone. Pellet sepharose and transfer lysate to
a new eppendorf tube
Add precipitating antibody at appropriate concentration for efficient recovery and incubate at 4
degrees for 1.5-2 hours
MRI is a medical imaging technique used in radiology to visualize detailed internal structures.
MRI uses no ionizing radiation instead it uses a powerful magnetic field to align the magnetization of
some atoms in the body, then uses radio frequency fields to systematically alter the alignment of this
magnetization. This causes the nuclei to produce a rotating magnetic field detectable by the scanner and
this information is recorded to construct an image of the scanned area of the body.
In an MRI machine a radio frequency transmitter produce an electromagnetic field. The photons of this
field flip the spin of the aligned protons in the body. As the intensity and duration of application of the
field increase, more aligned spins are affected. After the field is turned off, the protons decay to the
original spin-down state and the difference in energy between the two states is released as a photon. It
is these photons that produce the electromagnetic signal that the scanner detects. The frequency the
protons resonate at depends on the strength of the magnetic field.
MRI is used to image every part of the body, and is particularly useful for tissues with many hydrogen
nuclei and little density contrast, such as the brain, muscle, connective tissue and most tumors.
Applications
In clinical practice, MRI is used to distinguish pathologic tissue (such as a brain tumor) from normal
tissue. One advantage of an MRI scan is that it is harmless to the patient. It uses strong magnetic fields
and non-ionizing radiation in the radio frequency range, unlike CT scans and traditional X-rays, which
both use ionizing radiation.
While CT provides good spatial resolution (the ability to distinguish two separate structures an
arbitrarily small distance from each other), MRI provides comparable resolution with far better contrast
resolution (the ability to distinguish the differences between two arbitrarily similar but not identical
tissues). The basis of this ability is the complex library of pulse sequences that the modern medical MRI
scanner includes, each of which is optimized to provide image contrast based on the chemical sensitivity
of MRI.
Risks
MRI contains no ionizing radiation. To date, there have been no documented significant side effects
of the magnetic fields and radio waves used on the human body.
The most common type of contrast (dye) used is gadolinium. It is very safe. Allergic reactions to the
substance rarely occur. The person operating the machine will monitor your heart rate and
breathing as needed.
MRI is usually not recommended for acute trauma situations, because traction and life-support
equipment cannot safely enter the scanner area and the exam can take quite a bit of time.
People have been harmed in MRI machines when they did not remove metal objects from their
clothes or when metal objects were left in the room by others.
A gill is a respiratory organ found in many aquatic organisms that extracts dissolved oxygen from water
and excreting carbon dioxide. (It does not break up water molecules into hydrogen and oxygen to
absorb oxygen.) The gills of some species such as hermit crabs have adapted to allow respiration on land
and kept them moist. The microscopic structure of a gill presents a large surface area to the external
environment.
Many microscopic aquatic animals, and some that are larger but inactive, can absorb adequate oxygen
through the entire surface of their bodies, and so can respire adequately without a gill. However, more
complex or more active aquatic organisms usually require a gill or gills.
Gills usually consist of thin filaments of tissue, branches, or slender tufted processes that have a highly
folded surface to increase surface area. A high surface area is crucial to the gas exchange of aquatic
organisms as water contains only 1/20 the dissolved oxygen that air does.
With the exception of some aquatic insects, the filaments and lamellae (folds) contain blood or coelomic
fluid, from which gases are exchanged through the thin walls. The blood carries oxygen to other parts of
the body. Carbon dioxide passes from the blood through the thin gill tissue into the water. Gills or gill-
like organs, located in different parts of the body, are found in various groups of aquatic animals,
including mollusks, crustaceans, insects, fish, and amphibians.
Gills in Invertebrate
Respiration in the Echinodermata (includes starfish and sea urchins) is carried out using a very
primitive version of gills called papulae. These thin protuberances on the surface of the body contain
diverticula of the water vascular system.
Crustaceans, molluscs, and some insects have gills that are tufted or plate-like structures at the
surface of the body.
The gills of other insects are tracheal, and also include both thin plates and tufted structures, and, in
the larval dragon fly, the wall of the caudal end of the alimentary tract (rectum) is richly supplied
with tracheae as a rectal gill. Water pumped into and out of the rectum provides oxygen to the
closed tracheae.
Aquatic insects use a tracheal gill, which contains air tubes. The oxygen in these tubes is renewed
through the gills.
Physical gills
Physical gills are a type of structural adaptation common among some types of aquatic insects, which
holds atmospheric oxygen in an area with small openings called spiracles. The structure (often called a
plastron) typically consists of dense patches of hydrophobic setae on the body, which prevent water
entry into the spiracles. The physical properties of the interface between the trapped air bubble and
The physical gill mechanism allows aquatic insects with plastrons to remain constantly submerged.
Examples include many beetles in the family Elmidae, aquatic weevils, and true bugs in the family
Aphelocheiridae.
Gill in Pila:
Pila shows both aquatic and arial mode of respiration. The animal possesses the gill or ctendium for
aquatic respiration and pulmonary sac or lung for arial respiration. The left and right nuchal lobes are
the accessory structure.
Ctendium: It is a comb like structure present in branchial chamber. It consists of long ctendial axis
containing an efferent and an afferent blood vessel. The afferent carries deoxygenated and afferent
carries oxygenated blood from the heart to the gills and gill to heart respectively.
The axis bear long series of flat leaflets triangular shaped lamellae. Two sides of lamellae are unequal.
The short side receives blood from afferent vessel and is thus called afferent side and the other side is
called efferent side.
The middle lamellae are larger and decreased in size towards the two ends. Histologically lamellae form
a double layer of epithelium consists of non-ciliated columnar cells, ciliated columnar cells and glandular
cells.
Pulmonary Sac: It is a bag like structure developed from the mantle wall open by a pulmonary
aperture. Dorsal wall is pigmented while ventral wall are creamy white. These walls are muscular and
consist of blood spaces.
Nuchal Lobes: These are highly contractile structures of mantle present either side of head. The two
nuchal lobes form a sort of drain for entry and exit of water in and out of mantle cavity.
Mechanism of Aquatic Respiration: The two nuchal lobe form channel like structure. Water enters
in ospharidum, crossing over the epitenia, enters into branchial chamber, after washing the gills go out
from the right nuchal lobe.
Mechanism of Aerial Respiration: Left nuchal lobe exert and form a tube like structure called siphon.
The air enters in the pulmonary sac by siphon through pulmonary aperture. Air flow is maintained by
expansion and contraction of pulmonary sac. Wall of pulmonary sac contain blood vessels where
gaseous exchange take place.
Some invertebrates have "lungs" that serve a similar respiratory purpose as, but are not evolutionarily
related to, vertebrate lungs. Some arachnids have structures called "book lungs" used for atmospheric
gas exchange. The Coconut crab uses structures called Branchiostegal lungs to breathe air and indeed
will drown in water, hence it breathes on land and holds its breath underwater. The Pulmonata are an
order of snails and slugs that have developed "lungs".
The oldest book lungs have been recovered from extinct trigonotarbid arachnids preserved in the 410
million year old Rhynie chert of Scotland. These Devonian fossil lungs are almost indistinguishable from
the lungs of modern arachnids.
The absence or presence of book lungs divides the Arachnida into two main groups, the pulmonate
arachnids (book lungs present; scorpions and the Tetrapulmonata; whip scorpions, Schizomida,
Amblypygi, and spiders), and the apulmonate arachnids (book lungs absent; microwhip scorpions,
harvestmen, Acarina, pseudoscorpions, Ricinulei and sunspiders). One of the long-running controversies
in arachnid evolution is whether the book lung evolved once in the arachnid common ancestor, or
whether it evolved in multiple groups of arachnids in parallel as they came onto land.
The pulmonary chamber filled with the vertical folds called lamellae running parallel and arranged
over one another like the leaves of the book.
Each lamella is a hollow structure made up of two layers of cuticle united at their edges. A thin air space
is bounded in between two adjacent lamellae. The roof of the atrial chamber is perforated by many
linear slit like openings set parallel to each other. The atrial chamber communicates with the inter-
laminar air spaces through these openings.
The exchange of gases take place between the air of interlamellar space and the venous blood through
the thin membranous walls of the lamellae, the blood become oxygenated and of the air and is CO2
passed into the air.
Branchiostegal lung
A branchiostegal lung is a respiration organ used by some air-breathing arthropods. It is one of the most
significant adaptations of some crabs and hermit crabs such as coconut crabs to their terrestrial
habitats.
The branchiostegal (gill) tissue is supported by folds or other mechanisms to increase surface area and is
of a similar tissue to that normally found in gills. In this case, the lung is more suited to the absorption of
oxygen from air, rather than water.
Pulmonata
The Pulmonata, or "pulmonates," are an informal group (previously an order, and before that a subclass)
of snails and slugs characterized by the ability to breathe air, by virtue of having a pallial lung instead of
a gill, or gills. The group includes many land and freshwater families, and several marine families.
Pulmonates have a single auricle and kidney, and a concentrated, symmetrical, nervous system. The
mantle cavity is located on the right side of the body, and lacks gills, instead being converted into a
Instead of branchiostegal lungs, some terrestrial hermit crabs (Coenobita and Birgus) possess multiple
gills and small lungs, with other varieties of gas diffusion methods supporting the transition from aquatic
to terrestrial dwelling.
The developmental shift from water diffusion "gills" to air perfusion "lungs" may have been related to
the need for reduce rates of water loss in air.
Trachea
The invertebrate trachea refers to the open respiratory system composed of spiracles, tracheae, and
tracheoles that terrestrial arthropods have evolved to transport metabolic gases to and from tissues.
The distribution of spiracles can vary greatly among the many orders of insects, but in general each
segment of the body can have no more than one pair of spiracles, each of which connects to an atrium
and has a relatively large tracheal tube behind it. The tracheae are invaginations of the cuticular
exoskeleton that branch (anastomose) throughout the body with diameters from only a few
micrometres up to 0.8 mm. The smallest tubes, tracheoles, penetrate cells and serve as sites of diffusion
for water, oxygen, and carbon dioxide. Gas may be conducted through the respiratory system by means
of active ventilation or passive diffusion. Unlike vertebrates, insects do not generally carry oxygen in
their haemolymph. This is one of the factors that may limit their size.
A tracheal tube may contain ridge-like circumferential rings of taenidia in various geometries such as
loops or helices. In the head, thorax, or abdomen, tracheae may also be connected to air sacs. Many
insects, such as grasshoppers and bees, which actively pump the air sacs in their abdomen, are able to
control the flow of air through their body. In some aquatic insects, the tracheae exchange gas through
the body wall directly, in the form of a gill. Note that despite being internal, the tracheae of arthropods
are shed during moulting (ecdysis).
Some terrestrial woodlice have evolved pseudotrachea, also called corpora allata (singular: corpus
allatum), which is made up of air tubes that delivers oxygen to their haemolymph; a similar system has
been found in some caterpillars.
Structure of Tracheae:
In case of cockroach consists of a network of branched closed tubes called trachea lying within the
haemocoel. There is three pair of large parallel longitudinal tracheal trunks. One dorsal, one ventral
and one lateral in position. These are connected together by transverse commissure.
The tracheal trunk communicate to outside by spiracles or stigma. Each spiracle is surrounded by an
annular sclerite called peritreme and lead into a chamber called atrium from which the trachea
arises.
Mechanism:
Alternate contraction and relaxation of the abdominal muscle is cause rhythmic contraction and
expansion of the abdomen. Such movement causes change in the diameter of trachea and force air I and
out of the tracheal tubes through the spiracles. Oxygen reaches the tissue by diffusion through the wall
of the tracheoles.
Respiratory Pigments
A respiratory pigment is a molecule, such as hemoglobin in humans that increases the oxygen-carrying
capacity of the blood. The four most common invertebrate respiratory pigments are hemoglobin,
haemocyanin, haemerythrin and chlorocruorin. Hemoglobin is bright red when oxygenated, and dark
red when deoxygenated, oxygenated haemocyanin is blue in color, deoxygenated is almost colorless.
Oxygenated chlorocruarin turns green where oxygenated haemeryhrtin is a violet to pink colour, and
colorless when deoxygenated.
Hemoglobin
Hemoglobin and hemoglobin-like molecules are also found in many invertebrates, fungi, and plants. In
these organisms, hemoglobins may carry oxygen, or they may act to transport and regulate other
species such as carbon dioxide, nitric oxide, hydrogen sulfide and sulfide.
The giant tube worm Riftia pachyptila showing red hemoglobin-containing plumes.
The structure of hemoglobins varies across species. Hemoglobin occurs in all kingdoms of organisms, but
not in all organisms. Primitive species such as bacteria, protozoa, algae, and plants often have single-
globin hemoglobins. Many nematode worms, molluscs, and crustaceans contain very large multisubunit
molecules, much larger than those in vertebrates. In particular, chimeric hemoglobins found in fungi and
giant annelids may contain both globin and other types of proteins.
One of the most striking occurrences and uses of hemoglobin in organisms is in the giant tube worm
(Riftia pachyptila, also called Vestimentifera), which populates ocean volcanic vents. Instead of a
digestive tract, these worms contain a population of bacteria constituting half the organism's weight.
The bacteria react with H2S from the vent and O2 from the water to produce energy to make food from
H2O and CO2. The worms end with a deep red fan-like structure ("plume"), which extends into the water
and absorbs H2S and O2 for the bacteria, and CO2 for use as synthetic raw material similar to
photosynthetic plants. The structures are bright-red due to their containing several extraordinarily
complex hemoglobins that have up to 144 globin chains, each including associated heme structures.
These hemoglobins are remarkable for being able to carry oxygen in the presence of sulfide, and even to
carry sulfide, without being completely "poisoned" or inhibited by it as hemoglobins in most other
species are.
Hemocyanins (also spelled haemocyanins) are respiratory proteins in the form of metalloproteins
containing two copper atoms that reversibly bind a single oxygen molecule (O2). Oxygenation causes a
color change between the colorless Cu (I) deoxygenated form and the blue Cu (II) oxygenated form.
Hemocyanins carry oxygen in the hemolymph of most molluscs, and some arthropods, including the
horseshoe crab, Limulus polyp emus. They are second only to hemoglobin in biological popularity of use
in oxygen transport. Unlike the hemoglobin in red blood cells found in vertebrates, hemocyanins are not
bound to blood cells but are instead suspended directly in the hemolymph.
Catalytic activity
It is interesting to compare hemocyanin to the phenol oxidases (e.g. tyrosinase), homologous enzymes
sharing its type 3 Cu active site coordination. Hemocyanin also exhibits phenol oxidase activity, but with
slowed kinetics from greater steric bulk at the active site. Partial denaturation actually improves
hemocyanin’s phenol oxidase activity by providing greater access to the active site.
Explanation
Although the respiratory function of hemocyanin is similar to that of hemoglobin, there are a significant
number of differences in its molecular structure and mechanism. Whereas hemoglobin carries its iron
atoms in porphyrin rings (heme groups), the copper atoms of hemocyanin are bound as prosthetic
groups coordinated by histidine residues. Species using hemocyanin for oxygen transportation are
commonly crustaceans living in cold environments with low oxygen pressure. Under these
circumstances hemoglobin oxygen transportation is less efficient than hemocyanin oxygen
transportation.
Hemerythrin
Hemerythrin is an oligomeric protein responsible for oxygen (O2) transport in the marine invertebrate
phyla of sipunculids, priapulids, brachiopods, and in a single annelid worm, magelona. Recently,
hemerythrin was discovered in methanotrophic bacterium Methylococcus capsulatus. Myohemerythrin
is a monomeric O2-binding protein found in the muscles of marine invertebrates. Hemerythrin and
myohemerythrin are essentially colorless when deoxygenated, but turn a violet-pink in the oxygenated
state.
Chlorocruorin
Oxygen is weaker than that of most hemoglobin. A dichromatic compound, chlorocruorin is noted for
appearing green in dilute solutions, though it appears light red when found in concentrated solutions.
Its structure is very similar to erythrocruorin (which is likewise very similar to multiple subunits of
myoglobin) and it contains many 16-17kDa myoglobin-like subunits arranged in a giant complex of over
a hundred subunits with interlinking proteins as well with a total weight exceeding 3600kDa. Because of
its giant macromolecule structure, chlorocruorin is free floating in blood. The only significant difference
between chlorocruorin and erythrocruorin is that chlorocruorin carries an abnormal heme group
structure. This enormous macromolecule is typically found free floating in the plasma, and not
contained within red blood cells.
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures.
It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates
the analyte to be measured from other molecules in the mixture based on differential partitioning
between the mobile and stationary phases. Subtle differences in a compound's partition coefficient
result in differential retention on the stationary phase and thus changing the separation.
History
The history of chromatography begins during the mid-19th century. Chromatography, literally "color
writing", was used—and named— in the first decade of the 20th century, primarily for the separation of
plant pigments such as chlorophyll. New types of chromatography developed during the 1930s and
1940s made the technique useful for many types of separation process.
Some related techniques were developed during the 19th century (and even before), but the first true
chromatography is usually attributed to Russian botanist Mikhail Semyonovich Tsvet, who used columns
of calcium carbonate for separating plant pigments during the first decade of the 20th century during his
research of chlorophyll.
Chromatography became developed substantially as a result of the work of Archer John Porter Martin
and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and
basic techniques of partition chromatography, and their work encouraged the rapid development of
several types of chromatography method: paper chromatography, gas chromatography, and what would
become known as high performance liquid chromatography. Since then, the technology has advanced
rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many
different ways, resulting in the different varieties of chromatography described below. Simultaneously,
advances continually improved the technical performance of chromatography, allowing the separation
of increasingly similar molecules.
Chromatography Terms
Paper Chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that
are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink
experiments. Two-way paper chromatography, also called two-dimensional chromatography, involves
using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures
of similar compounds, for example, amino acids.
The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the
solvent molecules to the paper. As the solvent rises through the paper it meets and dissolves the sample
mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the
sample mixture travel at different rates due to competition between the paper fibers and solvent for the
solutes. Since paper is composed of cellulose, a polar substance, polar substances have a high affinity for
the paper. Paper chromatography takes anywhere from several minutes to several hours.
In some cases, paper chromatography does not separate pigments completely; this occurs when two
substances appear to have the same values in a particular solvent. In these cases, two-way
chromatography is used to separate the multiple-pigment spots
Ascending Chromatography
In this method, the solvent is set at the bottom of the vessel in which the paper is dipped supported by a
glass rod. The solvent is move in upward direction by the capillary action against the gravitational force.
Rƒ Value
The retention factor (Rƒ) may be defined as the ratio of the distance travelled by the solute to the
distance travelled by the solvent. If Rƒ value of a solution is zero, the solute remains in the stationary
phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and
travels with the solvent front.
Affinity Chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific
biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor
and ligand.
Principle
The immobile phase is typically a gel matrix, often of agarose which is a linear sugar molecule derived
from algae. The molecule of interest will have a well-known and defined property which can be
exploited during the affinity purification process. The target molecule is trapped on a solid or stationary
phase or medium. The other molecules in solution will not become trapped as they do not possess this
property. The solid medium can then be removed from the mixture, washed and the target molecule
released from the entrapment in a process known as elution. Possibly the most common use of affinity
chromatography is for the purification of recombinant proteins.
Alternatively binding may be achieved using a batch treatment, by adding the initial mixture to the solid
phase in a vessel, mixing, separating the solid phase , removing the liquid phase, washing, re-
centrifuging, adding the elution buffer, re-centrifuging and removing the eluate.
A third method, expanded bed adsorption, which combines the advantages of the two methods
mentioned above, has also been developed. The solid phase particles are placed in a column where
liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensures that
the solid phase does not exit the column with the liquid phase.
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer
chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin
layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose. This layer of adsorbent is
known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent
mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different
analytes ascend the TLC plate at different rates, separation is achieved.
Technique
The process is similar to paper chromatography with the advantage of faster runs, better separations,
and the choice between different stationary phases. Because of its simplicity and speed TLC is often
used for monitoring chemical reactions and for the qualitative analysis of reaction products.
A small spot of solution containing the sample is applied to a plate, about 1.5 centimeters from the
bottom edge. The solvent is allowed to completely evaporate off, otherwise a very poor or no
separation will be achieved. If a non-volatile solvent was used to apply the sample, the plate needs
to be dried in a vacuum chamber.
A small amount of an appropriate solvent in poured in a separation chamber. The TLC plate is then
set in it. The container is closed with a cover. The solvent moves up the plate by capillary action,
meets the sample mixture and carries it up the plate.
Analysis
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
Ion-Exchange Chromatography
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based
on their charge. It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually
separated components are called analytes. It is often used in protein purification, water analysis, and
quality control.
Principle
Ion exchange chromatography retains analyte molecules on the column based on ionic interactions. The
stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite
charge. This type of chromatography is further subdivided into cation exchange chromatography and
anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the
anionic species B- can be retained by the stationary phase.
Cation exchange chromatography retains positively charged cations because the stationary phase
displays a negatively charged functional group:
Anion exchange chromatography retains anions using positively charged functional group:
Typical Technique
A sample is introduced into a sample loop of known volume. A buffered aqueous solution known as the
mobile phase carries the sample from the loop onto a column that contains some form of stationary
phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with
covalently bonded charged functional groups. The target analytes (anions or cations) are retained on the
stationary phase but can be eluted by increasing the concentration of a similarly charged species that
will displace the analyte ions from the stationary phase. The analytes of interest must then be detected
by some means, typically by conductivity or UV/Visible light absorbance.
Uses
This technique is used for the separation of proteins.
Reversed-phase chromatography (RPC) includes any chromatographic method that uses a non-polar
stationary phase.
Stationary Phases
Any inert non-polar substance that achieves sufficient packing can be used for reversed-phase
chromatography. The most popular column is octadecyl carbon chain bonded silica with 297 columns
commercially available.
Elution can be performed isocratic (the water-solvent composition does not change during the
separation process) or by using a gradient (the water-solvent composition does change during the
separation process). The pH of the mobile phase can have an important role on the retention of an
analyte and can change the selectivity of certain analytes. Charged analytes can be separated on a
reversed phase column by the use of ion-pairing (also called ion-interaction). This technique is known as
reversed phase ion-pairing chromatography.
HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase and
analyte through the column, and a detector that provides a characteristic retention time for the analyte.
With HPLC, a pump provides the higher pressure required to propel the mobile phase and analyte
through the densely packed column. The increased density arises from smaller particle sizes. This allows
for a better separation on columns of shorter length when compared to ordinary column
chromatography.
Operation
The sample to be analyzed is introduced in small volume to the stream of mobile phase. The solution
movement through the column is slowed by specific chemical or physical interactions with the
stationary phase present within the column. The velocity of with the solution moves depends on the
nature of the sample and on the compositions of the stationary phase. The time at which a specific
Gas chromatography
Gas chromatography (GC), is a common type of chromatography used in analytic chemistry for
separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC
include testing the purity of a particular substance, or separating the different components of a mixture
(the relative amounts of such components can also be determined). In some situations, GC may help in
identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds
from a mixture
In gas chromatography, the mobile phase is a carrier gas, usually an inert gas such as helium or an
unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an
inert solid support, inside a piece of glass or metal tubing called a column. The instrument used to
perform gas chromatography is called a gas chromatograph.
The gaseous compounds being analyzed interact with the walls of the column, which is coated with
different stationary phases. This causes each compound to elute at a different time, known as the
retention time of the compound. The comparison of retention times is what gives GC its analytical
usefulness.
GC Analysis
In a GC analysis, a known volume of gaseous or liquid analyte is injected into the "entrance" (head) of
the column, usually using a micro syringe. As the carrier gas sweeps the analyte molecules through the
column, this motion is inhibited by the adsorption of the analyte molecules either onto the column walls
or onto packing materials in the column. The rate at which the molecules progress along the column
depends on the strength of adsorption, which in turn depends on the type of molecule and on the
stationary phase materials. Since each type of molecule has a different rate of progression, the various
components of the analyte mixture are separated as they progress along the column and reach the end
of the column at different times (retention time). A detector is used to monitor the outlet stream from
the column. Thus the time at which each component reaches the outlet and the amount of that
component can be determined. Generally, substances are identified (qualitatively) by the order in which
they emerge (elute) from the column and by the retention time of the analyte in the column.
Physical Components
Autosamplers
The autosampler provides the means to introduce a sample automatically into the inlets. Manual
insertion of the sample is possible but is no longer common. Automatic insertion provides better
reproducibility and time-optimization.
Columns
The tubing is usually made of stainless steel or glass and contains a packing of finely divided, inert, solid
support material that is coated with a liquid or solid stationary phase. The nature of the coating material
determines what type of materials will be most strongly adsorbed. Thus numerous columns are available
that are designed to separate specific types of compounds.
Detectors
A number of detectors are used in gas chromatography. The most common are the flame ionization
detector (FID) and the thermal conductivity detector (TCD). Both are sensitive to a wide range of
components, and both work over a wide range of concentrations. While TCDs are essentially universal
and can be used to detect any component other than the carrier gas (as long as their thermal
conductivities are different from that of the carrier gas, at detector temperature), FIDs are sensitive
primarily to hydrocarbons, and are more sensitive to them than TCD. However, an FID cannot detect
water. Both detectors are also quite robust. Since TCD is non-destructive, it can be operated in-series
before an FID (destructive), thus providing complementary detection of the same analytes.
Application
In general, substances that vaporize below ca. 300 °C (and therefore are stable up to that
temperature) can be measured quantitatively. The samples are also required to be salt-free; they
should not contain ions. Very minute amounts of a substance can be measured, but it is often
required that the sample must be measured in comparison to a sample containing the pure,
suspected substance.
Various temperature programs can be used to make the readings more meaningful; for example to
differentiate between substances that behave similarly during the GC process.
Professionals working with GC analyze the content of a chemical product, for example in assuring
the quality of products in the chemical industry; or measuring toxic substances in soil, air or water.
GC is very accurate if used properly and can measure picomoles of a substance in a 1 ml liquid
sample, or parts-per-billion concentrations in gaseous samples.
Electrophoresis
Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially
uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss,
who noticed that the application of a constant electric field caused clay particles dispersed in water to
migrate. It is ultimately caused by the presence of a charged interface between the particle surface and
the surrounding fluid.
Principle
The dispersed particles have an electric surface charge, on which an external electric field exerts an
electrostatic Coulomb force. According to the double layer theory, all surface charges in fluids are
screened by a diffuse layer of ions, which has the same absolute charge but opposite sign with respect
to that of the surface charge. The electric field also exerts a force on the ions in the diffuse layer which
has direction opposite to that acting on the surface charge. This latter force is not actually applied to the
particle, but to the ions in the diffuse layer located at some distance from the particle surface, and part
of it is transferred all the way to the particle surface through viscous stress. This part of the force is also
called electrophoretic retardation force.
In the olden days, DNA fragments were laboriously separated by using gravity. In the 1970s, the
powerful tool called DNA gel electrophoresis was developed, in which electricity was used to separate
DNA fragments by size as they migrate through a porous gel matrix. Gel electrophoresis is now used to
sort out strands of DNA, RNA or protein molecules by size, by using agarose gel and electrical current.
This is achieved by moving negatively charged DNA molecules through an agarose matrix to which an
electric field is applied (electrophoresis). Shorter molecules move faster and migrate farther than the
longer ones, thus separating them by size. DNA Gel electrophoresis is generally used after amplification
of DNA using PCR technique. It is done in the following steps:
To make agarose gel, put a little agarose into a flask and add buffer solution to it. The buffer aids
the electric current to travel through the gel. Heat the solution until agarose melts in the buffer and
then pours the material into the gel mould. Place the comb in mould to make wells in the gel in
which DNA can later be placed. When the gel solidifies, carefully remove the comb.
Pour buffer into the electrophoresis box. The buffer conducts the electric current and keeps the gel
from drying out.
Place the gel mould inside the electrophoresis box, barely submerged in the buffer.
Add loading buffer that contains dye to the DNA sample.
Use micropipette to load DNA sample mixed with the loading buffer into the first well of the agarose
gel.
Use micropipette to load DNA size standard into the next well of the agarose gel. The DNA size
standard contains strands of known length.
Connect the electrophoresis box to the electric current. The negative (black) end of the current
must be placed on the end closest to the wells.
Turn on the electric current. The DNA strands move away from the negative current. The short
strands move through the gel more quickly than the long strands. This can be observed due to the
blue dye of the loading buffer.
Turn off current and remove the gel mould from the electrophoresis box.
Place gel in DNA staining solution, ethidium bromide. This binds to DNA and can be viewed under
fluorescent light.
Remove gel from ethidium bromide after about 30 minutes and place it on UV light box. The DNA
bands will be visible for both the DNA sample and DNA size standard.
Precipitation is the formation of a solid in a solution or inside another solid during a chemical reaction or
by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or
when compacted by a centrifuge, a pellet. The liquid remaining above the solid is in either case called
the supernate or supernatant. Powders derived from precipitation have also historically been known as
flowers.
Natural methods of precipitation include settling or sedimentation, where a solid form over a period of
time due to ambient forces likes gravity or centrifugation. During chemical reactions, precipitation may
also occur particularly if an insoluble substance is introduced into a solution and the density happens to
be greater (otherwise the precipitate would float or form a suspension). With soluble substances,
precipitation is accelerated once the solution becomes supersaturated.
In solids, precipitation occurs if the concentration of one solid is above the solubility limit in the host
solid, due to e.g. rapid quenching or ion implantation, and the temperature is high enough that diffusion
can lead to segregation into precipitates. Precipitation in solids is routinely used to synthesize
nanoclusters.
An important stage of the precipitation process is the onset of nucleation. The creation of a hypothetical
solid particle includes the formation of an interface, which requires some energy based on the relative
surface energy of the solid and the solution. If this energy is not available, and no suitable nucleation
surface is available, supersaturation occurs.
Applications
Precipitation reactions can be used for making pigments, removing salts from water in water treatment,
and in classical qualitative inorganic analysis.
Precipitation is also useful to isolate the products of a reaction during workup. Ideally, the product of
the reaction is insoluble in the reaction solvent. Thus, it precipitates as it is formed, preferably forming
pure crystals. An example of this would be the synthesis of porphyrins in refluxing propionic acid. By
cooling the reaction mixture to room temperature, crystals of the porphyrin precipitate, and are
collected by filtration:
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures,
used in industry and in laboratory settings. More-dense components of the mixture migrate away from
the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis.
Chemists and biologists may increase the effective gravitational force on a test tube so as to more
rapidly and completely cause the precipitate ("pellet") to gather on the bottom of the tube. The
remaining solution is properly called the "supernate" or "supernatant liquid". The supernatant liquid is
then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a
Pasteur pipette.
The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in
revolutions per minute (RPM) or g. The particles' settling velocity in centrifugation is a function of their
size and shape, centrifugal acceleration, the volume fraction of solids present, the density difference
between the particle and the liquid, and the viscosity.
Basis of separation:
Size
Shape
Density
Methodology:
Utilizes density difference between the particles/macromolecules and the medium in which
these are dispersed
Dispersed systems are subjected to artificially induced gravitational fields
Differential centrifugation is a common procedure in microbiology and cytology used to separate certain
organelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is
first homogenized to break the cell membranes and mix up the cell contents. The homogenate is then
subjected to repeated centrifugations, each time removing the pellet and increasing the centrifugal
force. Finally, purification may be done through equilibrium sedimentation, and the desired layer is
extracted for further analysis.
Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal
forces. As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g
for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force
and greater times to pellet. In general, one can enrich for the following cell components, in the
separating order in actual application:
Sample preparation
Before differential centrifugation can be carried out to separate different portions of a cell from one
another, the tissue sample must first be homogenized. In this process, a blender, usually a piece of
porous porcelain of the same shape and dimension as the container is used. The container is, in most
cases, a glass boiling tube.
The tissue sample is first crushed and a buffer solution is added to it, forming a liquid suspension of
crushed tissue sample. The buffer solution is a dense, inert, aqueous solution which is designed to
suspend the sample in a liquid medium without damaging it through chemical reactions or osmosis. In
most cases, the solution used is sucrose solution; in certain cases brine will be used. Then, the blender,
connected to a high-speed rotor, is inserted into the container holding the sample, pressing the crushed
sample against the wall of the container.
With the rotator turned on, the tissue sample is ground by the porcelain pores and the container wall
into tiny fragments. This grinding process will break the cell membranes of the sample's cells, leaving
individual organelles suspended in the solution. This process is called homogenization. A portion of cells
will remain intact after grinding and some organelles will be damaged, and these will be catered for in
the later stages of centrifugation.
Since different fragments of a cell have different sizes and densities, each fragment will settle into a
pellet with different minimum centrifugal forces. Thus, separation of the sample into different layers can
be done by first centrifuging the original homogenate under weak forces, removing the pellet, then
exposing the subsequent supernatants to sequentially greater centrifugal fields. Each time a portion of
different density is sedimented to the bottom of the container and extracted and repeated application
produces a rank of layers which includes different parts of the original sample. Additional steps can be
taken to further refine each of the obtained pellets.
Sedimentation depends on mass, shape, and partial specific volume of a macromolecule, as well as
solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored during the
experiment to calculate molecular weight. Values of sedimentation coefficient (S) can be calculated.
Large values of S (faster sedimentation rate) correspond to larger molecular weight. Dense particle will
sediments more rapidly. Elongated proteins have larger frictional coefficients and sediment more slowly
to ensure accuracy.
Equilibrium sedimentation
Equilibrium sedimentation uses a gradient of a solution such as caesium chloride or sucrose to separate
particles based on their individual densities (mass/volume). It is used as a purifying process for
differential centrifugation. A solution is prepared with the densest portion of the gradient at the
bottom. Particles to be separated are then added to the gradient and centrifuged. Each particle
proceeds (either up or down) until it reaches an environment of comparable density. Such a density
gradient may be continuous or prepared in a stepped manner. For instance, when using sucrose to
prepare density gradients, one can carefully float a solution of 40% sucrose onto a layer of 45% sucrose
and add further less dense layers above. The homogenate, prepared in a dilute buffer and centrifuged
briefly to remove tissue and unbroken cells, is then layered on top. After centrifugation typically for an
hour at about 100,000 x g, one can observe disks of cellular components residing at the change in
density from one layer to the next. By carefully adjusting the layer densities to match the cell type, one
can enrich for specific cellular components. Caesium chloride allows for greater precision in separating
particles of similar density. In fact, with a caesium chloride gradient, DNA particles that have
incorporated heavy isotopes (13C or 15N for example) can be separated from DNA particles without
heavy isotopes.
Flow cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles,
such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an
electronic detection apparatus. It allows simultaneous multipara metric analysis of the physical and/or
chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in
the diagnosis of health disorders, especially blood cancers, but has many other applications in both
research and clinical practice. A common variation is to physically sort particles based on their
properties, so as to purify populations of interest.
Principle
A beam of laser light of a single wavelength is directed onto a hydrodynamically-focused stream of fluid.
A number of detectors are aimed at the point where the stream passes through the light beam: one in
line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and
one or more fluorescent detectors). Each suspended particle passing through the beam scatters the ray
and may be excited into emitting light at a longer wavelength than the light source. This combination of
scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness
at each detector, it is then possible to derive various types of information about the physical and
chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on
the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic
granules or the membrane roughness). Some flow cytometers on the market have eliminated the need
for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each
cell's fluorescence, scattered light, and transmitted light.
Density gradient is a variation in density over an area. In the life sciences, a special technique called
density gradient separation is used for isolating and purifying cells, viruses and subcellular particles.
There are two stages of separation with centrifuges. The first stage takes advantage of the difference in
the terminal velocities of different particles as determined by Stoke's law:
Where vt is the terminal velocity of the particle, R the radius of the particle, a is the centrifugal
acceleration of the centrifuge, µ the viscosity of the medium, ps the density of the particle, and p the
density of the medium.
From the above equation, it is apparent that the terminal velocity is a function of the particle radius and
density. Thus, on the average, bigger and heavier particles will travel through the medium faster and
settle at the bottom of a centrifuge tube in a shorter time. Because a typical mixture of cell homogenate
contains organelles of varying sizes and densities, as well as shapes, they can be separated according to
the sedimentation speed. The decrease in the sedimentation time in a centrifuge over gravitational
settling is mainly due to the considerable increase in the variable in Stoke's equation.
In the study of cell organelles, different fractions of the subcellular particles are routinely separated with
a centrifuge. It was by this type of isolation technique that mitochondria were discovered to be
responsible for the entire tricarboxylic acid cycle and ribosomes accountable for protein biosynthesis.
In a typical separation scheme, an isotonic sucrose solution is mixed with cells. The mixture is placed in a
bead homogenizer and the cell membranes are broken to spill out the cell contents. The resulting
mixture of subcellular organelles can be placed in a centrifuge and spun at 1000g for 10 minutes. Intact
cells and heavy nuclei are collected at the bottom of the centrifuge tube as a pellet. After the
supernatant is further centrifuged at 10,000g for 20 minutes, subcellular particles of intermediate
terminal velocities such as mitochondria, lysosomes, and microbodies may be collected. The smaller and
lighter particles (ribosomes, endoplasmic reticulum fragments, cell membranes, and microsomes) can be
further separated from the supernatant of the preceeding stage by centrifugation at 100,000g for 60
minutes. The final supernatant may be considered to be the soluble portion of the cell cytoplasm. Notice
Each fraction obtained through differential centrifugation contains quite a few different types of
organelles which have similar sedimentation velocities, i.e. similar values of R2 (ps-p). Because this
factor is a combination of both the size and the density, the fraction can be further separated based on
density alone irrespective of the sizes. This second stage can be accomplished by a process known as
density gradient centrifugation.
Honey Bee, common name for any of several species of highly social bees known for their honey-
hoarding behavior and their use as a domesticated species. The European honey bee is important in
modern agriculture and in nature, providing pollination for many valuable crops and wild plants.
Social Organization
The honey bee is a social insect that can survive only as a member of a community, or colony. The
colony inhabits an enclosed cavity, its nest. Domesticated colonies are kept in artificial containers,
usually wooden boxes, known as hives.
Castes
The honey bee community consists of three structurally different forms-the queen (reproductive
female), the drone (male), and the worker (nonreproductive female). These castes are associated with
different functions in the colony; each caste possesses its own special instincts geared to the needs of
the colony.
The Queen
The queen is the only sexually productive female in the colony and thus is the mother of all drones,
workers, and future queens. Her capacity for laying eggs is outstanding; her daily output often exceeds
1500 eggs, the weight of which is equivalent to that of her own body.
Anatomically, the queen is strikingly different from the drones and workers. Her body is long, with a
much larger abdomen than a worker bee. Her mandibles, or jaws, contain sharp cutting teeth, whereas
her offspring have toothless jaws. The queen has a curved, smooth stinger that she can use repeatedly
without endangering her own life. In contrast, the worker honey bees are armed with straight, barbed
stingers, so that when a worker stings, the barbed, needlesharp organ remains firmly anchored in the
flesh of its victim. In trying to withdraw the stinger, the bee tears its internal organs and dies shortly
thereafter. The queen bee lacks the working tools possessed by worker bees, such as pollen baskets,
beeswax-secreting glands, and a well-developed honey sac. Her larval food consists almost entirely of a
secretion called royal jelly that is produced by worker bees. The average lifespan of the queen is one to
three years.
As with all bees, pollen is the principal source of protein, fat, minerals, and vitamins, the food elements
essential for the growth and development of larvae of all three castes. Adult bees can subsist on honey
or sugar, a pure carbohydrate diet. Besides gathering and storing food for all the members of the colony,
the workers are responsible for maintaining the brood at 33.9? C (93? F), the optimum temperature
required for hatching the eggs and rearing the young. When the nest or hive becomes too hot the
workers collectively ventilate it by fanning their wings. During cool weather, they cluster tightly about
the nursery and generate heat. The eggs, which are laid one per cell, hatch in three days. The larvae are
fed royal jelly for at least two days and then pollen and nectar or honey. Each of the hundreds of larvae
in a nest or hive must be fed many times a day.
For the first three weeks of their adult lives, the workers confine their labors to building the honeycomb,
cleaning and polishing the cells, feeding the young and the queen, controlling the temperature,
evaporating the water from the nectar until it thickens as honey, and many other miscellaneous tasks.
At the end of this period, they function as field bees and defenders of the colony. The workers that
develop early in the season live extremely busy lives, which, from egg to death, last about six weeks.
Worker bees reared late in the fall usually live until spring, since they have little to do in the winter
except eat and keep warm. Unlike other species of bees, honey bees do not hibernate; the colony
survives the winter as a group of active adult bees.
Drones are prevalent in colonies of bees in the spring and summer months. As fall approaches, they are
driven out of the nests or hives by the workers and left to perish.
As eusocial insects, termites live in colonies that, at maturity, number from several hundred to several
million individuals. Colonies use decentralised, self-organised systems of activity guided by swarm
intelligence to exploit food sources and environments that could not be available to any single insect
acting alone. A typical colony contains nymphs (semi-mature young), workers, soldiers, and reproductive
individuals of both genders, sometimes containing several egg-laying queens. Termites are sometimes
called "white ants", though they are not closely related to true ants.
Cast system
Termites have following casts
Reproductives
Reproductives possess compound eyes and are more or less brown due to their sclerotized cuticle.
Developing reproductives have wing buds, wings or wing stumps. Reproductives can be further divided
into:
Alates: The young winged reproductives of both sexes. From time to time about 100 to 1000 alates
leave the colony for a mating and colonising flight. After mating a pair settles down at a suitable site
like a rotting scar on a tree in order to establish a new colony.
De-alates: Alates that cast their wings after the colonising flight and successively turn into queens
and kings are called as de-alates. Initially only a few eggs are laid and brought up by a female de-
alate. As the number of individuals in the colony grows, the more workers are available to help the
young queen to care for the brood. After three to five years the number of individuals is already so
large, that the colony of a pest species can turn into the damaging stage.
Queen and king: Which are the main reproductive individuals in a colony? Once there are many
workers to help the queen, her only job is to produce a tremendous number of offspring. A large
queen may lay more than 1000 eggs per day. The life span of a queen can be as much as 50 years.
Neotenics: Neotenics assist the queen in laying eggs, once her productivity decreases. When the
queen has died or deteriorated, one of the neotenics takes her place. That is the reason why the
removal of a queen from her colony does not necessarily mean the end of the colony.
Workers
Workers are sterile, wingless and blind males and females. Their cuticle is unpigmented and not
hardened; therefore the animals are confined to a dark and moist environment. Workers build the nest
and galleries, they fetch food, care for the brood and feed reproductives and soldiers. The worker’s life
span is one to two years.
Economic Importance
Due to their wood-eating habits, many termite species can do great damage to unprotected buildings
and other wooden structures. Their habit of remaining concealed often results in their presence being
undetected until the timbers are severely damaged and exhibit surface changes. Once termites have
entered a building, they do not limit themselves to wood; they also damage paper, cloth, carpets, and
other cellulosic materials. Particles taken from soft plastics, plaster, rubber, and sealants such as silicone
rubber and acrylics are often employed in construction.
Ants
Workers: These are sterile females possessing reduced thorax, small gaster and small eyes
Soldiers: These are sterile females with large heads and powerful mandibles
Queen: These are fertile females with large gaster
Aners: These are slender fertile females provided with two pairs of wings
Ants mostly construct underground nests. The female lay eggs following mating in nuptial flight and
after mating the males die
Biomembranes
Molecular composition and arrangement functional consequences.
Transport across cell membrane-Diffusion, active transport and pumps, uniports, symports and
antiports
Membrane potential
Co-transport by symporters or anti-porters
Transport across epithelia
Cytoskeleton
Microfilaments and microtubules-structure and dynamics
Microtubules and mitosis
Cell movements-intracellular transport, role and kinesin and dynein, signal, transduction
mechanisms.
Cell-Cell signaling
Cell surface receptors
Second messenger system
MOP kinase pathways
Signaling from plasma membrane to nucleus
Cell cycle
Cyclines and cycline dependent kinases
Genome organization
Hierarchy in organization
Mobile DNA
Uptake into ER
Trafficking mechanisms.
Biology of cancer
Biology of aging
Glycolysis, citric acid cycles its regulation and role as metabolic hub
Cholesterol biosynthesis, its metabolism steroid genesis, Bile acids and their metabolism derrayed
cholesterol level
Primary, Second, tertiary and quaternary structure of proteins (Domain, Reverse turn of
Ramachandran plot)
Ultra centrifuge
NMR spectrophotometer
Microscopes
Microbiological Techniques:
Media preparation and sterilization
Cryotechniques:
Cryopreservation for cells, tissue, organisms
Autoradiography
Metabolic labeling
Biosensors
Cytotaxonomy
Molecular taxonomy
Methodology
Taxonomic collections, preservation, curetting process and identification
Association index
Measures of Dispersion:
Range
Interquartile range
Quartile deviation
Mean deviation
Standard deviation
Correlation:
Methods studying correlation
Scatter diagram method
Graphic method
Karl Pearson coefficient of correlation
Rank correlation.
Chi-square analysis
Analysis of variance
Computer in Biometrics
Components of computers
Use of Microsoft (Word, Excel, Power Point)
Use of Microsoft Excel for statistical calculation and graphical representation of data
Surfing through Internet
Organization of coelom
Pseudocoelomates
Metamerism in Annelida
Locomotion
Flagella and ciliary movement in Protozoa
Respiration
Organs of respiration: Gills, lungs and trachea
Respiratory pigments
Mechanism of respiration
Minor Phyla
Concept and significance
Excretion
Organs of excretion
Coelom, coelomoducts
Nephridia
Malphigian tubules
Mechanism of excretion
Nervous system
Primitive nervous system: Coelenterata and Echinodermata
Advanced nervous system: Annelida, Arthropoda (Crustacean and Insecta) and Mollusca
(Cephalopoda)
Mouthparts of Insects
Metamorphosis in insects
Invertebrate larvae
Larval forms of free living invertebrates
Taxonomy (from Greek from the words taxis = order and nomos = law) may refer to either a hierarchical
classification of things, or the principles underlying the classification. Taxonomy is the science of
classifying organisms. Taxonomy Includes
Biosystematics
Biosystematics is the science through which life forms are discovered, identified, described, named,
classified and catalogued, with their diversity, life histories, living habits, roles in an ecosystem, and
spatial and geographical distributions recorded. In essence, it is biosystematics, the science that
provides indispensable information to support many fields of research and beneficial applied programs.
Biological systematics is the study of the diversification of life on the planet Earth, both past and
present, and the relationships among living things through time. Relationships are visualized as
evolutionary trees (synonyms: cladograms, phylogenetic trees, phylogenies). Phylogenies have two
components, branching order (showing group relationships) and branch length (showing amount of
evolution). Phylogenetic trees of species and higher taxa are used to study the evolution of traits (e.g.,
anatomical or molecular characteristics) and the distribution of organisms (biogeography). Systematics,
in other words, is used to understand the evolutionary history of life on Earth.
"Systematic biology" and "taxonomy" (terms that are often confused and used interchangeably) were
defined in relationship to one another as follows:
The term "systematics" is sometimes used synonymously with "taxonomy" and may be confused with
"scientific classification". However, Taxonomy is more specifically the identification, description, and
naming (i.e. nomenclature) of organisms while "classification" is focused on placing organisms within
hierarchical groups that show their relationships to other organisms. All of these biological disciplines
can be involved with extinct and extant organisms.
Elements of Taxonomy
Taxon
A taxon (plural: taxa) is a group of (one or more) organisms, which a taxonomist adjudges to be a unit.
Usually a taxon is given a name and a rank, although neither is a requirement. A taxon is a taxonomic
unit, a group of organisms that has been named.
Taxonomists sometimes make a distinction between "good" (or natural) taxa and others that are "not
good" (or artificial). Today it is common to define a good taxon as one that reflects presumptive
evolutionary (phylogenetic) relationships. But this is not mandatory.
A taxon may be given a formal name, a scientific name. Such a scientific name is governed by one of the
Nomenclature Codes, which sets out rules to determine which scientific name is correct for that
particular grouping.
A taxonomic unit, whether named or not: i.e. a population, or group of populations of organisms which
are usually inferred to be phylogenetically related and which have characters in common which
differentiate the unit (e.g. a geographic population, a genus, a family, an order) from other such units. A
taxon encompasses all included taxa of lower rank and individual organisms.
Ranks
A taxon can be assigned a rank, usually (but not necessarily) when it is given a formal name. The rank of
a given taxon is not necessarily fixed, but can be altered later by another (or the same) taxonomist.
"Phylum" applies formally to any biological domain, but traditionally it was always used for animals,
whereas "Division" was traditionally often used for plants, fungi, etc.
Superclass
Class
Subclass
Infraclass
In biological classification, rank is the level (the relative position) in a taxonomic hierarchy. Examples of
taxonomic ranks are species, genus, family, and class. Each rank subsumes under it a number of less
general categories. The rank of species, and specification of the genus to which the species belongs is
basic, which means that it may not be necessary to specify ranks other than these. The International
Code of Zoological Nomenclature defines rank as:
The level, for nomenclatural purposes, of a taxon in a taxonomic hierarchy (e.g. all families are for
nomenclatural purposes at the same rank, which lies between superfamily and subfamily).
Categories
The second feature of Linnaeus's taxonomy system that simplifies organism classification is the ordering
of species into broad categories. There are seven major categories: Kingdom, Phylum, Class, Order,
Family, Genus, and Species.
A good aid for remembering these categories is the mnemonic device: Keep Plates Clean or Family Gets
Sick.
Some of these categories can be further divided into intermediate categories such as subphyla,
suborders, superfamilies, and superclasses. An example of this taxonomy scheme is: Kingdom
Phylum
Subphylum
Superclass
Class
Subclass
Superorder
Order
Suborder
Superfamily
Family
Subfamily
Genus
Subgenus
Species
Subspecies
The history of taxonomy stretches back to the 4th century BC, to the times of Aristotle and before. Since
the first classification systems emerged, dividing the world of life into various groups with various
relationships, scientists have grappled with the task of keeping classification in sync with scientific
evidence.
He was the father of biological classification. He said “Animals may be characterized according to their
way of living, their actions, their habits and their body parts”. He established numerous collective
categories using different characters like blooded Vs bloodless, two footed Vs four footed, hairy Vs
feathered, with or without an outer shell and so forth. Aristotle was among the first to document the
division of life forms into animals and plants. Aristotle classified animals according to observation, for
example, he defined high-level groups of animals by whether or not they had red blood (this roughly
reflects the division between vertebrates and invertebrates used today).
Plantae – plants
Animalia – animals
Specialization becomes more pronounced. More and more author becomes specialist of single
group like birds or beetles etc.
Classification becomes more hierarchical. Linnaeus recognized only kingdoms, classes, orders,
genera, and species but phylum and families was added to the classification scheme later
Philosophical guidelines were expressly rejected and the classifying become an entirely empirical
enterprise
The search for a natural system was intensified with the term natural serving as the antonym of
artificial
The three kingdom system, introduced by Ernst Haeckel in 1894, reflected the long-standing two
kingdoms (Plantae and Animalia) that can be attributed to Aristotle (perhaps before) and added third
kingdom, Protista that included single-celled eukaryotes and bacteria (prokaryotes).
The important changed introduced by this classification scheme was the introduction of the Kingdom
Bacteria. This reflected the growing understanding that bacteria (single-celled prokaryotes) were very
Robert Whittaker's 1959 classification scheme added a fifth kingdom to Copeland's four kingdoms, the
Kingdom Fungi (single and multi-cellular osmotrophic eukaryotes)
In 1977, Carl Woese extended Robert Whittaker's Five Kingdoms to replace Kingdom bacteria with two
kingdoms, Eubacteria and Archaebacteria. Archaebacteria differ from Eubacteria in their genetic
transcription and translation processes (in Archaebacteria, transcription and translation more closely
resembled eukaryotes). These distinguishing characteristics were shown by molecular genetic analysis.
In 1990, Carl Woese put forth a classification scheme that greatly overhauled previous classification
schemes. The three-domain system he proposed is based on molecular biology studies, and resulted in
the placement of organisms into three domains.
Bacteria
Archaea
Eukarya
Applications of Biosystematics
In Theoretical Biology
Population thinking has come into biology through the taxonomy and taxonomy is the root of the
population genetics.
The problem of multiplication of species was solved by taxonomist.
Taxonomy made the greatest contribution to the understanding of the structure of species and the
evolutionary role of peripheral population.
Taxonomy upholds the importance of natural selection when early mendelions thought that
mutation had eliminated the role of natural selection as an evolutionary factor.
H.W Botes and F. Muller made significant contribution to the understanding of mimicry and related
evolutionary phenomenon.
Taxonomist and naturalist in close contact with the taxonomy were instrumental in the
development of the ethology and the study of the phylogeny of behaviour.
Taxonomist plays an important role in counteracting the reductionist tendencies dominant in so
much of functional biology and contribute to a healthy balance in biological science.
In applied biology
The contribution of taxonomy to the applied biology includes medicines, public health,
agriculture, conservation, management of natural resources.
It supplies the key to the solution of previously perplexing problems in economic entomology. For
example the famous case of epidemiology of malaria. The malaria mosquito anopheles
maculipennis meigen is reported throughout the country Europe. Yet malaria was restricted to
the certain local districts the large amount of money was wasted because no one understands
the connection between the distribution of the mosquito and that of malaria. Careful taxonomic
studied finally provided the key. The maculipennis complex was found to consist of several
subling species with different habitat preferences and breeding habits, only some of which are
Chemotaxonomy
Chemotaxonomy (from chemistry and taxonomy), also called chemosystematics, is the attempt to
classify and identify organisms (originally plants), according to demonstrable differences and similarities
in their biochemical compositions. The compounds studied in most of the cases are mostly proteins,
amino acids and peptides. Examples of chemotaxonomic markers are phospholipid-derived fatty acids
and enzymes.
Chemotaxonomy also called chemosystematics is the attempt to classify and identify organism by
chemical means. It deals with the structure and function of cellular components such as proteins,
carbohydrates, lipids, nucleic acids and other biomolecules. The compounds studied in most of the cases
are mostly protein.
Chemical Technique: A suitable technique is chosen for chemical survey depends upon the
group to be investigated.
Comparative Study: A critical comparative study of collected data lead to some acceptable
interpretation.
Chemical Characters:
Directly Visible Characters: Serology provides the earliest used methods of comparing
proteins. This method is based on the fact that the protein of one organism will show a stronger
antibody reaction to the protein of a closely related organism rather than distinctly related.
Proteins: Protein for example hemoglobin and fibirinopeptide of man and chimpanzee show
similarity thus established close relationship among them.
Cytotaxonomy
The utilization of cytological characters for explaining the taxonomic problems and classification is called
as the cytotaxonomy. The following cytological characters are used:
Chromosome Number: The chromosome number is species specific and can use as a taxonomic
tool. E.g. the phylogeny of horses, Asses and zebra has been classified by their chromosome
number.
Chromosome Size: Size of chromosome is varies from species to species so can use as a taxonomic
tool. E.g. the orthoptera has large chromosome and diptera have giant chromosome i.e. salivary
chromosome.
Chromosome Morphology: The chromosome may be of single armed of double armed. Also the
position of centromere differs in different cases. Additionally the chromosomes may be of equal or
different lengths. These all facts can use as a taxonomic tool.
The use of molecular constituents explaining the taxonomic problems and classification is called as the
molecular taxonomy.
The proteins, nuclear DNA, mitochondrial DNA and ribosomal nucleic acids are used as taxonomic
tool. For example:
The study of restriction site mapping of ribosomal DNA established a relation in genus Rana.
The rRNA has been particularly useful for an understanding of the relationship of the lower
eukaryotes and various branches of prokaryotes.
The sequencing of protein and nucleic acid also provide large amount of information by comparing
the sequence of base pairs of two homologous DNA or RNA. For Example:
The hemoglobin and fibirinopeptide of man and chimpanzee show similarity thus established
close relationship among them.
The technique of DNA hybridization attempts to determine the total overall similarity of two taxa at
molecular level.
Techniques like chromatography and electrophoresis are used to study the taxonomy of specific
chemical components and macromolecules.
Species Concept
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is
often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in
many cases this definition is adequate, more precise or differing measures are often used, such as
similarity of DNA, morphology or ecological niche. Presence of specific locally adapted traits may further
subdivide species into subspecies. There are following species concepts are there:
There are strict limits to the possible variation within any one species
Disadvantages
Give no idea about speciation
Give no idea about any developing variation through time
Concept doesn’t really work with evolution
Give no idea about ‘type’ characteristics
Give no idea about the true identity of the archetype
Disadvantages
Give no idea about sympatric species, morphologically indistinguishable, but are clearly different
lineages? e.g., Bacillus species form identical colonies, live in the same environment, but are
totally distinct according to sequence data.
Cryptomorphic sibling species: where substantial genetic differences exist, but are not manifest
in morphology
Give no idea about sexual dimorphism: where male and female morphologies differ
Disadvantages
Difficult to recognize, because many organisms occupy different niches due to adaptation or
developmental changes
Does the ecological species concept provide much resolving power for microbes?
Disadvantages
This concept does not account for genomic hybrids, where genes have passed from one taxon to
another, and the genetic make-up of individuals can be traced to different phylogenies or
genealogies
Completely sequenced microbial genomes have demonstrated that such hybrids are common:
5-15% of one bacterial species’ genomes can be attributed to acquisition from other species,
making the evolutionary species concept practically irrelevant to prokaryotes.
Phenotypic clusters based on mainstream bacteria systematics. It is based on numerical taxonomy and
analogous to morphological species concept, but adds physiology to anatomy.
This concept integrates character-based concepts that emphasize the presence of an apparent
organismal attribute with history based concepts that emphasize the degree of relatedness of a new
isolate to previously characterized organisms
By constructing a complete set of phylogenetic trees derived from proteome databases, the
phylogenomic approach facilitates the inclusion of horizontal genetic exchange events in the
identification of microbial species (Ogenseitan 2002).
Polytypic species (Rassenkreis): Rensch (1929) proposed the German term Rassenkreis (which literally
means "Circle of races" for those species, which have two or more geographically isolated interbreeding
populations.
Sibling species:
They are true sympatric species that are morphologically identical or nearly so but are reproductively
isolated. For example, Drosophila pseudo-obscura and D. persimilis are sibling species.
Subspecies:
This term was used by Schlegel in 19th century as a replacement for variety. Subspecies is a
geographically isolated (allopatric) population of a species that differs morphologically from other
populations of the species but does not exhibit reproductive isolation. A trinomen is used to designate a
subspecies. For example, Cervus elephus elephus is the continental red deer and Cervus elephus
scoticus is the red deer of Great Britain.
Deme:
This is a minimal interbreeding local population unit of a species which share a single gene pool. Demes
live in most suitable areas that are separated by regions of unsuitable conditions and are affected by the
gene flow from adjacent areas.
Cline:
This is an evolutionary concept proposed by J.H. Huxley (1939) and is defined as a gradation in
measurable characters. Cline is formed by a series of contiguous populations in which a given character
changes gradually. Two opposite ends of the series may be very different but difficult to be called
subspecies due to the absence of geographical isolation. The terms geocline (geographic), ecocline
(ecological) and chronocline (succession lines) are also used.
Race:
This is not a recognized taxonomic category. They are local populations of a species which are affected
by the local conditions and therefore develop morphological differences for example human races.
In biology, races are distinct genetically divergent populations within the same species with relatively
small morphological and genetic differences. The populations can be described as ecological races if
they arise from adaptation to different local habitats or geographic races when they are geographically
isolated. If sufficiently different, two or more races can be identified as subspecies, which is an official
biological taxonomy unit subordinate to species. If not, they are denoted as races, which means that a
formal rank should not be given to the group, or taxonomists are unsure whether or not a formal rank
should be given. According to Ernst W. Mayr, "a subspecies is a geographic race that is sufficiently
different taxonomically to be worthy of a separate name". Examples of race include:
Hypodigm:
All specimens personally known to taxonomists at the time of describing species and used by him
collectively as a sample on which his inferences are based upon.
Subspecies (commonly abbreviated subsp. or ssp.) in biological classification is either a taxonomic rank
subordinate to species or a taxonomic unit in that rank (plural: subspecies). A subspecies cannot be
recognized in isolation: a species will either be recognized as having no subspecies at all or two or more,
never just one.
The differences between subspecies are usually less distinct than the differences between species, but
more distinct than the differences between races or breeds. The characteristics attributed to subspecies
generally have evolved as a result of geographical distribution or isolation.
Members of one subspecies differ morphologically or by different coding sequences of a peptide from
members of other subspecies of the species. Subspecies are defined in relation to species.
If the two groups do not interbreed because of something intrinsic to their genetic make-up (perhaps
green frogs do not find red frogs sexually attractive, or they breed at different times of year) then they
are different species.
If, on the other hand, the two groups would interbreed freely provided only that some external barrier
were removed (perhaps there is a waterfall too high for frogs to scale, or the populations are far distant
from one another) then they are subspecies. Other factors include differences in mating behavior or
time and ecological preferences such as soil content.
A monotypic species has no distinct population or races, or rather one race comprising the whole
species. Monotypic species can occur in several ways:
All members of the species are very similar and cannot be sensibly divided into biologically significant
subcategories.
The individuals vary considerably but the variation is essentially random and largely meaningless so far
as genetic transmission of these variations is concerned.
Classification is the groping of the objects into classes owning to their shared possession of characters.
Rules of classification:
Items that are to be classified are assembled into classes that are made as homogenous as
possible
An individual item is included in that class with the members of which It share the greatest
number of attributes
A separate class is established for any item that is too different to be included in one of the
previously established classes
The degree of difference among the classes is expressed by arranging them in a hierarchy of
nested sets. Each category level in the hierarchy expresses a certain level of distinctions.
Functions of classification:
Classification is serving as the identification key for newly discovered species
Classification is an index to store information like books in library
A good Classification has the heuristic properties. It permits one to make prediction concerning
the attributes of other member of taxon including those still undiscovered and previously
unused characters
Classification permits the making of generalizations. The validity of generalization derived from
comparisons depends on the Classification on which the comparison is based. The comparative
method is most reliable when homogeneous classes are compared.
A Classification has explanatory power
Analogy: It is phenotypic similarity. It is not related to the community of descents but is due to
the similarity of functions.
Convergence: The acquisition of a similar character by two taxa whose common ancestor
lacked that character is called as convergence.
Parallel Characters: These are the similar characters derived independently by related taxa
with a similar genetic background.
Theories of Classification
In biology, one main method used to classify species into groups is the phenetic, or phenotypic, method.
The phenetic method group’s species according to their observable phenetic attributes: if two species
look more like each other than either does to any other species, they will be grouped together. The full
classification consists of a taxonomic hierarchy of levels, such that the members of different groups at
higher and higher levels look decreasingly similar to one another. For example, a wolf and a dog (same
genus) look phenetically more alike than do a wolf and a dolphin which are in the same Class. Nothing
needs to be known about evolution in order for species to be classified phenetically.
Phenetics employ four criteria to determine whether or not a given classification is satisfactorily
represent the probable relationship of the included taxa:
A classification is considered more natural when is based on more characters. Phenetics based
on large number of characters so considered more natural.
Phenetics results the phenogram. A phenogram however is not a classification but several
methods have been suggested to convert a phenogram into a classification.
Use of data matrix permitted rapid and accurate comparison of the similarities and differences
of various taxa. It also encouraged the search for as many differentiating characters as possible.
The phenetic system is useful when the determination of degree of similarity is the objective.
Disadvantages of Phenetics
Practical difficulties
To assemble large data to make a data matrix in phenetics is a tedious and time consuming task.
All the characters in phenetics have equal weight so important characters are diluted in all other
rudiment characters.
A phenetic classification cannot be improved gradually because any addition of new characters
requires a new analysis.
This method lacks the theoretical justification of choice among clustering method. Different
people use different method for clustering and hence results in erroneous result.
In phenetic classification the general idea has been to try to classify organisms in such a way
that organisms that are similar to each other are placed together in the same pool. The general
problem is that it is potentially at odds with the idea of trying to reflect ancestry.
There is a lack of objectivity in the phenetic system. The phenetic classification represents the
morphological similarity for large numbers of characters. Any consistency in a classification of
this sort does not follow from the phenetic system itself; there is no hierarchy of morphological
similarity which exists in nature. A phenetic classification is imposed subjectively by the
taxonomist.
The amount of unweighted phenetic similarity reflects an equal amount of underlying similarity
of the genotype and can be safely used for ranking.
The use of a sufficiently large number of characters makes it unnecessary to partition them into
similarities due to descent and those due to homoplasy.
Theoretical difficulties
Strict empiricism: It rejected the theory of evolution.
Failure to take mosaic evolution into account: Different parts of body, different stages of life
cycle and different type of characters tend to have different evolutionary rates. As a
consequence it will lead to different similarities estimates if either one character is used. This
happens in phenetics because the ignorance of arbitrary character.
Since no distinction is made in phenetics between primitive and derived characters. Pheneticists
should not able to distinguish between ancestral and derived groups.
The results obtained from phenetics allow for no testing, no meaningful prediction and no
interpretation of the nature of the achieved classification.cv
Phylogenetic Vs Phenetic
Only entities that have evolutionary relations can be classified in phylogenetic system. In contrast to the
phenetic principle, it classifies species according to how recently they share a common ancestor. Two
species that share a more recent common ancestor will be put in a group at a lower level than two
species sharing a more distant common ancestor.
For instance the wolf and the dog opposite share a more recent common ancestor than do a wolf and a
dolphin: they are therefore both classified in the genus Canis.
As the common ancestor of two species becomes more and more distant, they are grouped further and
further apart in the classification. In the end, all species are contained in the category of the set of all
living things which contains all the descendants of the most distant common ancestor of life.
Cladistics Vs Phenetic
A phenetic classification could be used to classify geological specimens: stone, rock, things that have not
evolved by biological processes. Cladism can only be used when things have an actual ancestral history -
like living things, possibly like human languages.
Hening proposed a method of classification that he called phylogenetic systematics which is exclusively
based on the genealogy i.e. on the branching pattern of phylogeny. This method was based on the one
component of phylogeny i.e. the branching points of lineages, the second component of phylogeny i.e.
relative subsequent divergence, is lacking in this system. To prevent confusion with phylogeny this
methodology is renamed as cladistics.
It is a method of analysis (genetic analysis, biochemical analysis, morphological analysis) that determines
relationships between organisms that are based solely on their evolutionary history.
Cladistics (also called phylogenetic or phylogenetic systematics) looks to the evolutionary history of
organisms to form the underlying framework for their classification. Cladistics therefore differs from
phenetics in that it is based on phylogeny (the evolutionary history of a group or lineage), not on the
observation of physical similarities.
When a species splits during evolution it will usually form two descendant species, called sister
species, and in a cladistics classification sister species are grouped together.
Monophyletic groups contain the common ancestor and all of its descendants.
Paraphyletic groups, descended from a common ancestor.
Polyphyletic groups, the results of convergent evolution.
Synapomorpy, a homologous character shared by two or more taxa and inferred to have been
present in the nearest common ancestor but not in earlier ancestor nor in taxa outside this
group.
Apomorphy, A derived character state.
Plesiomorphy, An ancestral character state.
Transformation series, a series of homologous characters some of which were derived from
others during evolution.
Holophyletic or Monophyletic, pertaining to a group that consist all the decedents of its most
recent common ancestor.
When a new group originates by hybridization between two species then the shape of the
phylogeny does not imply a hierarchical classification.
It give methods to weight the character like the weight is given to characters those derived from
nearest common ancestor and those retained from more remote ancestor.
Cladism has a deep philosophical justification that phenetic systems lack. Cladism is objective,
and objective classifications are preferable to subjective ones.
Despite theoretical advantages, cladism can run into practical problems. The uncertainties of
phylogenetic inference make cladistic classifications liable to frequent revision.
Disadvantages of Cladism
Cladistic classifications are natural for cladistic characters, but may be artificial for non-cladistic
characters.
A strictly cladistic classification could theoretically have an impractically large number of levels.
If we could count all the branch points between the earliest common ancestor of all modern
species and the present, the number would be ludicrously high, requiring far more levels than
the existing Linnaean system.
Only synapomorphic characters are used to ranking taxa. The plesiomorphic characters are
ignored.
Autapomorphic characters are those evolve in only one of two sister groups and these
characters are also ignored.
Ancestor descendent relationship is one of the important source of information on the relative
amount of differences among taxa are almost completely ignored by cladists when they
construct the classification.
Cladogram
To characterizing the evolutionary history of a group of organisms, scientists develop this tree-like
diagram called cladogram. The branching pattern revealed by cladistic analysis is represented by the
cladist in a diagram called as cladogram which is based on the pattern of synapomorphies.
Each branching point in a cladogram represents a speciational event which potentially gives rise
to a new holophytic taxon.
No branch of cladogram can be based on the plesiomorphic character.
Sister groups are placed at the same distance from the ancestral species and the sister taxon
that has retained more primitive character is placed on the left branch of the dichotomy.
These diagrams consist of a series of branches and leaves that represent the evolution of groups
of organisms through time.
A cladogram ignores all the anagenetic changes (The accumulation of changes in ancestor to
descendent lineages) that are not synapomorphic.
A natural classification is one in which the members of a group resemble one another not only in the
characters that define the group (as they must, by definition) but also for many other non-defining
characters too. This is opposed to an artificial classification in which the members of a group only
resemble each other in the defining characters; they show no similarities for non-defining characters
The advantage of natural classification is that it is possible to predict the distribution of other characters
from the classificatory groupings alone.
2. Classifications that mix more than one type of information are less informative than classifications
that represent only a single property. The problem with evolutionary classification is that it is
constructed with both phenetic and phylogenetic information: if you are given the classification, you do
not know which groups are phenetic and which phylogenetic.
Biological classification, or scientific classification in biology, is a method by which biologists group and
categorize organisms by biological type, such as genus or species. Biological classification is a form of
scientific taxonomy.
Degree of difference
Evolutionary role
Size of taxon
Stability
Analysis
Classification
Ranking of sister Same categorical rank Different rank if they differ by sufficient
groups autapomorhies
Stem group Always with crown group Sometimes with sister group
Species
The fundamental unit of taxonomy is the species. This is a group of very similar individuals that have the
potential to interbreed freely, to produce fertile offspring - but cannot interbreed successfully with
individuals of other species.
In paleontology however, it can never be known for certain whether a population with a particular
morphology was reproductively isolated or not. Hence, the definition of a fossil species (and most living
specimens) must be based almost entirely on morphological criteria. Sometimes, this can be
supplemented by a comparison of the chemistry of the shell, but only rarely.
The scientific name for a species consists of 2 parts - it is binomial, with a generic name followed by a
trivial (or specific) name.
Genus
The generic name refers to the genus, which is a group of species that are fairly closely related - such as
the genus Equus which includes several species, such as the Equus caballus, Equus asinus and Equus
zebra (domestic horse, wild ass and zebra respectively).
The generic name (Equus) can be used alone, to describe a genus, whereas the specific name is always
used with the generic name - it is meaningless when used alone.
The generic name always begins with a capital letter, and generic and species names are always printed
in italic (or underlined when writing or typing, when italic is not available).
Family
Genera are grouped into families, which are major groups of generally similar organisms; such as
Felidae, which includes all cat-like animals from domestic cat to wild lynx to tiger to cheetah to jaguar to
snow leopard.
Family names always end in the letters "ae", but are not printed in any special way.
However; all members vary significantly from the plant-eating animals, such as those in the major order
Artiodactyla, which includes the pigs, deer, giraffe and antelopes.
Orders begin with a capital and usually end in "a" - but not always, so it is not always easy to tell what is
an order!
Class
Orders are grouped into classes. The class is a major division within the animal Kingdom, and forms the
basis on which most fossil study is based.
Gastropoda
Cephalopoda
Pelecypoda
Phylum
Classes are grouped into phyla (the plural of phylum).
There are only about 30 phyla in the animal kingdom, and only about a dozen of these (including
Mollusca and Brachiopoda) leave any fossil remains. Thus, the vast majority of life has left no evidence
for us to find.
Within the animal Kingdom, Animalia, the most common phyla are:
Kingdom
Phyla are grouped into kingdom
A taxonomic character is any attribute of a member of a taxon by which it differs or may differ
from member of a different taxon.
A characteristic by which members of the two taxa agree but differ from member of third taxon
is a taxonomic character.
To define taxonomic character any attribute of organism is not correct. Features by which
organism of same taxa may differ in case of sex age and colour etc. this is not attribution of
taxonomic character.
Taxonomic character means population character.
Taxonomic character means actual or potential taxonomic difference such as “eyes red” versus
“eyes white”.
Thus taxonomic character means taxon A is differ from taxon B which universally accepted in
taxonomic literature of the last 200 years.
Complex structure has greater weight than simple structure. Complex ornamentation, complex
cusp pattern of teeth, and complex colour pattern all fall under this category.
Joint possession of derived characters has greater weight. Taxa can be defined on the basis of
shared derived characters and not of shared ancestral ones.
A character that is constant throughout the large group of species has greater weight than a
variable character.
A character that is present in one group and absent in other group will has greater weight.
A character that is not affected by ecological shift will has greater weight.
Proper classifying work is possible only adequate material of many species has available for comparison.
Museum provides this opportunity. So the taxonomist prefers those character which are easily observed
in preserved species. Taxonomic character can be divided into several kinds.
1. Morphological Characters
Feature of external morphology may vary according to the kinds of species. They range from such
superficial feature such as plumage and pelage character of birds and mammals through scale counts of
fish and reptiles to the highly conservative phylogenetically to significant sutures and sclerites of
anthropod body. The internal character can provide an abundant source of taxonomic character
practically all groups of animals. Morphological characters include the following terms:
General external morphology
Special Structures
Internal morphology
Embryology
Karyology and other cytological differences
Weighing of Character
Most taxa differ morphologically from other taxa. Typically, closely related taxa differ much less than
more distantly related ones, but there are exceptions to this. Cryptic species are species which look very
similar, or perhaps even outwardly identical, but are reproductively isolated. Conversely, sometimes
unrelated taxa acquire a similar appearance as a result of convergent evolution or even mimicry. A
further problem with relying on morphological data is that what may appear, morphologically speaking,
to be two distinct species, may in fact be shown by DNA analysis to be a single species.
2. Physiological Characters
This type of character is hard to define. All structures are the product of growth process that is of
physiological process thus ultimately physiological character. All physiology is controlled by enzyme and
other macromolecules. It includes:
Metabolic factor
Serological protein and other biochemical difference
Body secretion
Genic sterility factor
Immunological distances
Electrophoretic differences
Amino acid sequences of protein
DNA hybridization
DNA and RNA sequences
Restriction endonuclease analysis
4. Ecological Characters
It is now well established that every species has its nearest relative but food preference, tolerance to
other, ecological factor, resistance to predator competitors and other ecological factor. All these are to
the ecological character of fish. It includes:
5. Ethological Characters
Behavior is undoubtedly one of the most important sources of taxonomic character. Indeed behavioral
character is often inferior to morphological character in study of closely related species particularly
sibling species. Such as:
Weighing of Character
The behavioral character cannot be study in preserved specimen and behavior is intermittent even in
the living animal.
Certain behavior occurs during breeding seasons during a part of 24 hour period. The comparative study
of behavior of related species has become an autonomous discipline, Comparative ethnology.
Behavioral characteristic are important isolating mechanism for animals and new adaptation also
initiated by other behavior pattern.
6. Geographical Characters
The geographical characters are useful tools for clarifying confused taxonomic picture and for testing a
taxonomic hypothesis. It includes:
Taxonomic collection: Museums are the reservoirs of taxonomic collection. They supply a permanent
record of faunas and floras particularly for localities where biota has been destroyed.
Collection may need again a later period for the verification of original data.
Collections are the reference tools which are not in continuous use yet must be available when
needed.
Purpose of taxonomic collection: The adequate size material is required because:
More material is needed in a species with strong individuals and geographic variation than in a
uniform species.
More material is needed for studies of a specific or a subspecific character than for the studies
of the character of higher taxa.
The size of sample thus depends upon the objective of the research.
Where and how to collect: A collection trip must be planned carefully. Following point should be
kept in mind when data has to be collected:
All possible geographic information must be obtained beforehand, including the distribution of
vegetation types, altitudes and type of seasons.
Preservation of specimen: The specimens are preserved to make them to least subjected to
deterioration through the action of the:
Insect pets
Mold
The label should be written in the field where the specimen is prepared.
The label may contain information about the locality, date, collector name, sex, weight and
colour etc.
Identification: it involves:
Sorting of collections
Determination labels
Taxonomic Keys
In biology, an identification key is a printed or computer-aided device that aids the identification of
biological entities, such as plants, animals, fossils, microorganisms, and pollen grains. Identification keys
are also used in many other scientific and technical fields to identify various kinds of entities, such as
diseases, soil types, minerals, or archaeological and anthropological artifacts.
A taxonomic key is a device used by biologists for identifying unknown organisms. Keys are constructed
so that the user is presented with a series of choices about the characteristics of the unknown
organisms; by making the correct choice at each step of the key, the user is ultimately led to the identity
of a specimen. If each step has only two alternatives, the key is said to be dichotomous, else it is
polytomous. Modern multi-access or interactive keys allow the user to freely choose the identification.
Examining a specimen carefully and noting its characteristics before beginning to key it out are good
habits to develop when trying to identify plants. Listed below are some other helpful hints for the
successful use of taxonomic keys:
Read any introductory comments concerning the format of the key, abbreviations, and so forth,
before using the key.
Use a glossary to find the definition of any terms you do not understand.
Because living organisms are always somewhat variable, do not make a decision based on a
single specimen; instead, arrive at an average by examing several different specimens.
The character used at each identification step should be diagnostic; that is, each alternative
should be common to all members of a group of entities, and unique to that group. It should
also be differential, meaning that the alternatives should separate the corresponding subgroups
from each other.
Redundant characters should be used at each step. For example, if a group is to be split into two
subgroups, one characterized by six black spots and the other by four brown stripes, the user
should be queried about all three characters (number, shape, and color of the markings) — even
though any single one of them would be sufficient in theory. This redundancy improves the
reliability of identification, provides a consistency check against user errors, and allows the user
to proceed even if some of the characters could not be observed. In this case, the characters
should be ordered according to their reliability and convenience.
The terminology used in the identification steps should be consistent in meaning and should be
uniformly used. The use of alternative terms for the same concept to achieve more "lively
prose" should be avoided. Positive statements should be used in preference to negative
statements.
Geographic distribution characters should be used with caution. Species that have not been
observed in a region may still occasionally occur there. Also, the organism may have been
transported, particularly to locations near ports and airports, or it may have changed its range
(e. g., due to global warming).
Variant forms: The key may identify only some forms of the species, such as adult males
Incomplete coverage: Species and groups that are difficult to identify or that have been poorly
characterized may have been left out of the key, or may be mentioned only in introductory text.
Lighting and magnification: Very few keys give details of how the specimen was viewed (the
magnification, lighting system, angle of view etc.).
Language: Very few keys are multilingual. Translations of a key may be incorrect or misleading.
Many keys contain vague words that do not translate.
Indented key
Indented means slightly away from the margin of the page. The couplets in this key are indented from
the left hand margin of the margin of the page in such a way as to show their importance. Thus the two
members of primary couplets are near to left hand margin and secondary couplets are indented after
leaving 4-5 spaces and so on to the end of key.
Merits:
The relationship of the various divisions is apparent to the eye.
Demerits:
When the key size is long the alternative may be widely spread and results wasteful of space.
Bracket Key:
In this key the number to each couplet is given and backflow is possible by using the number present
inside bracket.
Merits:
The couplets are composed of alternatives placed side by side for ready comparison.
Demerits:
The relationship of the various divisions is not apparent to the eye.
Pictorial Key:
This key is uses the pictures and use for identification by nonscientist.
Taxonomic Publication
Mayr has discussed the nature of taxonomic publication under such heading as synopsis, review,
revision, catalog, monograph, atlas, fauna, manual, handbook and field guide.
Checklists: A checklist provides a convenient source of reference for the correct names of
specimens and the arrangement of collection. A list of names deserves to be called a checklist only if
a careful distinction is made within it between valid names and synonyms.
Revisions: These are taxonomic papers dealing with all the species of a species group, genus or
higher taxon. They provide a critical analysis of all nominal species and description of previously
undescribed species.
Monographs: These are complete systematic publications. They provide a full systematic
treatment of all the species, subspecies and other taxonomic units and require a thorough
knowledge on the part of the author of comparative anatomy, the biology of species and subspecies
included the immature stages in a group that exhibit metamorphosis and detailed distribution of
data.
Atlases: These publications provide complete illustrations of the species of a taxonomic group
reflecting the inadequacy of the printed word in conveying the mental picture of the general facies
of an animal.
Faunal works: In a fauna the animal of a circumscribed region are listed and described. In the case
of well-known organisms local fauna gives the local naturalist an opportunity to provide ecological
and behavioral information often based on the many years of fieldwork.
Evolutionary and biological publications: The author can publish his article in general journals
such as ecology, evolution or systematic zoology, where these articles will draw attention to the
monograph. Every volume of systematic zoology contain papers dealing with cytological or chemical
attributes of species, distribution pattern, behaviour, taxometrics, variability, endemism, extinction,
serology, geographic variation, speciation, rates of evolution, climatic rules, allometry and so on.
The International Code of Zoological Nomenclature (ICZN) is a widely accepted convention in zoology
which rules the formal scientific naming of organisms treated as animals. The rules, often referred to as
"the Code" (or "ICZN Code"), regulate principally
It was first published in 1961, although it has precedents going back to 1842; the 3rd edition was from
1985, the present edition is the fourth edition (effective since 2000). The Code is published in an English
and a French version, both versions are official and equivalent in force, meaning and authority.[2] This
means that if something in the English Code is unclear or its interpretation ambiguous, the French
version is decisive. The French Code is not online. The Code deals with zoological nomenclature, which is
defined in the Glossary as:
"The system of scientific names for animal taxa and the provisions for the formation, treatment, and use
of those names"
Principles of ICZN
Species have a name composed of two names, a "binomen": a generic name and a specific
name. No other rank can have a name composed of two names:
Giraffa camelopardalis
Subspecies have a name composed of three names, a "trinomen": generic name, specific name,
subspecific name:
Taxa at a rank above species have a name composed of one name, a "uninominal name".
Giraffa or Giraffidae
In botanical nomenclature, the equivalent for "binominal nomenclature" is "binary nomenclature" (or
sometimes "binomial nomenclature").
2. Principle of Coordination
It states that the act of publishing a new zoological name thereby automatically and simultaneously
establishes all the corresponding names in the relevant other ranks, with the same type.
In the species-group, publishing the species name (the binomen) Giraffa camelopardalis Linnaeus, 1758
also establishes the subspecies name (the trinomen) Giraffa camelopardalis camelopardalis Linnaeus,
1758. The same applies to the name of a subspecies; this establishes the corresponding species name.
In the genus-group, similarly, publishing the name of a genus also establishes the corresponding name
of a subgenus (or vice versa): Giraffa Linnaeus, 1758 and Giraffa Linnaeus, 1758. In the family-group,
publication of the name of a family, subfamily, superfamily (or any other such rank) also establishes the
names in all the other ranks in the family group.
4. Principle of Homonymy
It states that any one name, in one particular spelling, may be used only once (within its group). This will
be the first-published name; any later name with the same spelling (a homonym) is barred from being
used. The Principles of Priority and the First Reviser apply here. For family-group names the termination
(which is rank-bound) is not taken into account.
5. Principle of Priority
It states that the correct formal scientific name for an animal taxon, the name that is to be used, called
the valid name, is the oldest available name that applies to it. There are exceptions; another name may
be given precedence by any provision of the Code or by any ruling of the Commission.
It is the fundamental guiding precept that preserves the stability of zoological nomenclature. It was first
formulated in 1842 by a committee appointed by the British Association to consider the rules of
zoological nomenclature; the committee's report was written by Hugh Edwin Strickland.
6. Principle of Typification
It states that any named taxon, in the family group, genus group or species group, has (or should have) a
name-bearing type which allows the application of the name of the taxon to be objectively applied. The
type does not define the taxon; this is done by a taxonomist, and an indefinite number of competing
definitions can exist, side by side. Rather, a type is a point of reference; a name has a type, and a
taxonomist (having defined his taxon) can inventory which existing types fall within the scope of his
taxon. He or she can then use the rules in the Code to determine the valid name for the taxon.
Popular names are convenient for common usage; but they may be incomplete or duplicate - there are
often many names for one species, and a single name can be used for different species.
This name allows instant and exact placement of the organism within its taxonomic hierarchy (except
where errors and duplications have been made, of course!).
These names are always in Latin, or are "Latinised" when new words are used. They are at all times:
They are international, so identical whatever language is in use (as any names are either Latin, or
'Latinised')
If a new form is discovered, then its name is created by the worker who first published its description.
A major problem with classification is that it has, and is, changing. This is largely because of advanced
techniques of molecular sequencing, which give far more details than could ever be seen from a
complete organism.
Structure of ICZN
Family-group names
Genus-group names
Species-group names
The names above the family group are regulated only as to the requirements for publication; there is no
restriction to the number of ranks and the use of names is not restricted by priority.
The names in the family group, the genus group and the species group are fully regulated by the
provisions in the Code. There is no limitation to the number of ranks allowed in the family group. In the
genus group there are only two ranks: the genus and the subgenus. In the species group there are only
two ranks: the species and the subspecies.
Gender agreement
In the species group gender agreement applies. The name of a species, in two parts, a binomen, say,
Loxodonta africana, and of a subspecies, in three parts, a trinomen, say Canis lupus albus, originally is a
Latin phrase, and must be grammatically correct Latin. If the second part, the specific name (or the third
For instance, the generic name Equus is masculine; in the name Equus africanus the specific
name africanus is an adjective and its ending follows the gender of the generic name.
In Equus zebra the specific name zebra is a noun, it may not be "corrected" to "Equus zebrus".
In Equus quagga burchellii the subspecific name burchellii is a noun in the genitive ("of the
esteemed Burchell").
If a species is moved, therefore, the spelling of an ending may need to be changed. Confusion over
proper Latin grammar has led to many incorrectly-formed names appearing in print. An improper
automated search may fail to find all the variant spellings of a given name (e.g., the spellings atra and
ater may refer to the same species). Accordingly, many laymen and some scientists object to continued
adherence to this long-standing rule.
Homonym
In biology, a homonym (taxonomic homonym) is a name for a taxon that is identical in spelling to
another such name that belongs to a different taxon.
The rule in the International Code of Zoological Nomenclature is that the first such name to be
published is the senior homonym and is to be used (it is "valid"); any others are junior
homonyms and must be replaced with new names. It is, however, possible that if a senior
homonym is archaic, and not in "prevailing usage," it may be declared a nomen oblitum and
rendered unavailable, while the junior homonym is preserved as a nomen protectum.
Similarly, the International Code of Botanical Nomenclature specifies that the first published of
two or more homonyms is to be used: a later homonym is "illegitimate" and is not to be used
unless conserved.
Names those are similar enough that they are likely to be confused, are also considered to be
homonymous.
Both Codes only consider taxa that are in their respective scope (animals for the ICZN; primarily
plants for the ICBN). Therefore, if an animal taxon has the same name as a plant taxon, both
names are valid.
In zoological nomenclature, synonyms are different scientific names that pertain to the same taxon, for
example two names for the same species. The rule of zoological nomenclature is that the first name to
be published is the senior synonym; any others are junior synonyms and should not be used.
Objective synonyms unambiguously refer to the same taxon; this is the case if they refer to the same
description or the same type specimen or type species. Otherwise the synonyms are subjective
synonyms, meaning that there is room for debate: one researcher might consider the two (or more)
types or descriptions to refer to one and the same taxon, another might consider them distinct.
Objective synonyms are common at the level of genera, because for various reasons two genera may
obtain the same type species; these are objective synonyms. At the species level, subjective synonyms
are common because an unexpectedly large range of variation in a species, or simple ignorance about
an earlier description, may lead a biologist to describe a newly discovered specimen as a new species.
It is possible for a junior synonym to be given precedence over a senior synonym,[6] primarily when the
senior name has not been used since 1899, and the junior name is in common use. The older name
becomes a nomen oblitum, and the junior name is declared a nomen protectum. This is primarily to
prevent the confusion that would result if a well-known name, with a large accompanying body of
literature, were to be replaced by a completely unfamiliar name. Such a reversal of precedence is also
possible if the senior synonym was established after 1900, but only if the ICZN Commission approves an
application.
A nomen oblitum (Latin for "forgotten name") is a scientific name which is both a synonym and has
fallen into disuse by the scientific community.
In its present meaning, the nomen oblitum came into being with the fourth, 1999, edition of the
International Code of Zoological Nomenclature. After 1 January 2000, a scientific name may be formally
declared to be a nomen oblitum when it has not been used as a valid name within the scientific
community since 1899, and when it is either a senior synonym (there is also a more recent name which
applies to the same taxon, and which is in common use) or a homonym (it is spelled the same as another
name, which is in common use). Once it has formally been declared be a nomen oblitum, the disused
name is to be 'forgotten'. By the same act, the junior name must be declared to be a nomen protectum;
from then on, it takes precedence.
A type specimen is a vernacular term (not a formally defined term) typically used for an
individual or fossil that is any of the various name-bearing types for a species. For example, the
type specimen for the species Homo neanderthalensis was the specimen "Neanderthal-1"
discovered by Johann Karl Fuhlrott in 1856 at Feldhofer in the Neander Valley in Germany,
consisting of a skullcap, thigh bones, part of a pelvis, some ribs, and some arm and shoulder
bones. There may be more than one type specimen, but there is (at least in modern times) only
one holotype.
A type species is the nominal species that is the name-bearing type of a nominal genus or
subgenus.
A type genus is the nominal genus that is the name-bearing type of a nominal family-group
taxon.
The type series are all those specimens included by the author in a taxon's formal description,
unless the author explicitly or implicitly excludes them as part of the series.
Holotype: When a single specimen is clearly designated in the original description, this
specimen is known as the holotype of that species. The holotype needs to be placed in a major
museum, or similar well-known public collection, so that it is freely available for later
examination by other biologists.
Isotype: An isotype is a biological specimen duplicate of the holotype collected in the same
place and at the same time (in the type locality).
Paratype: Any additional specimen other than the holotype, listed in the type series, where
the original description designated a holotype. These are not name-bearing types.
Neotype: A specimen later selected to serve as the single type specimen when an original
holotype has been lost or destroyed, or where the original author never cited a specimen.
Syntype: Any of two or more specimens listed in a species description where a holotype was
not designated; historically, syntypes were often explicitly designated as such, and under the
present Code this is a requirement, but modern attempts to publish species description based
on syntypes are generally frowned upon by practicing taxonomists, and most are gradually being
replaced by lectotypes. Those that still exist are still considered name-bearing types.
Paralectotype: Any additional specimen from among a set of syntypes, after a lectotype has
been designated from among them. These are not name-bearing types.
Hapantotype: A special case in Protistans where the type consists of two or more specimens
of "directly related individuals representing distinct stages in the life cycle"; these are
collectively treated as a single entity, and lectotypes cannot be designated from among them.
Topotype: A specimen taken from the type-locality, which can sometimes also be a paratype,
neotype or lectotype.
Diversity index
A diversity index is a statistic which is intended to measure the differences among members of a set
consisting of various types of objects. Diversity indices can be used in many fields of study to assess the
diversity of any population in which each member belongs to a unique group, type or species. For
instance, it is used in ecology to measure biodiversity in an ecosystem, in demography to measure the
distribution of population of various demographic groups, in economics to measure the distribution over
sectors of economic activity in a region, and in information science to describe the complexity of a set of
information. Below, a series of diversity indices is discussed.
1. Species richness
The species richness S is simply the number of species present in an ecosystem. This index makes no use
of relative abundances. In practice, measuring the total species richness in an ecosystem is impossible,
except in very depauperate systems. The observed number of species in the system is a biased estimator
of the true species richness in the system, and the observed species number increases non-linearly with
sampling effort. Thus S, if indicating the observed species richness in an ecosystem, is usually referred to
as species density.
2. Species Evenness
The species evenness is the relative abundance or proportion of individuals among the species.
3. Concentration ratio
Concentration ratio is a crude indicator of the extent to which a few groups such as species,
demographic groups or companies dominate an environment, the total share taken by the top n species
or firms. However by itself the concentration ratio does not indicate how much that share is divided
between those top n firms or species.
With this index, 0 represents infinite diversity and 1, no diversity. That is, the bigger the value of D, the
lower the diversity. This is neither intuitive nor logical, so to get over this problem, D is often subtracted
from 1 to give:
The value of this index also ranges between 0 and 1, but now, the greater the value, the greater the
sample diversity. This makes more sense. In this case, the index represents the probability that two
individuals randomly selected from a sample will belong to different species.
Another way of overcoming the problem of the counter-intuitive nature of Simpson's Index is to take the
reciprocal of the Index:
The value of this index starts with 1 as the lowest possible figure. This figure would represent a
community containing only one species. The higher the value the greater will be the diversity. The
maximum value is the number of species (or other category being used) in the sample. For example if
there are five species in the sample, then the maximum value is 5.
As an example, let us work out the value of D for a single quadrat sample of ground vegetation in
woodland. Of course, sampling only one quadrat would not give you a reliable estimate of the diversity
of the ground flora in the wood. Several samples would have to be taken and the data pooled to give a
better estimate of overall diversity.
Then:
These 3 different values all represent the same biodiversity. It is therefore important to ascertain which
index has actually been used in any comparative studies of diversity. A value of Simpson's Index of 0.7 is
not the same as a value of 0.7 for Simpson's Index of Diversity.
Simpson's Index gives more weight to the more abundant species in a sample. The addition of rare
species to a sample causes only small changes in the value of D.
=2C / A + B
With this index 0 represents less similarity and 1 represents more similarity
=2C / A + B
=2 x 2 / 4 + 3
=0.57
D= n / N
D= dominance
Suppose a habitat has 110 species and number of an species ABC is 40 than
D= 40 / 110
= 2.7
Association Index
This statistical test attempts to measure the extent of linkage equilibrium within a population by
quantifying the amount of recombination among a set of sequences and detecting association between
alleles at different loci (Maynard-Smith et al., 1993). The Index of Association (IA) is calculated as
follows:
IA =VO/VE -1
If V0 is the observed variance of K and VE is the expected variance of K, where K is the number of loci at
which two individuals differ. If there is linkage equilibrium because of frequent recombination events,
the expected value of IA is zero. Clonal populations are identified by an IA value that differs significantly
from zero.