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FUNDAMENTALS OF CELL AND MOLECULAR BIOLOGY

Unit I

Cell introduction:

In biology, cell theory is the historic scientific theory and is universally accepted that all living
organisms are made up of ‘cells. The scientists have defined that the cells are the basic structural and
organizational unit of all living organisms, and that all cells originate from pre-existing cells.
Principally, the cells are the basic unit of structure in all organisms and also the basic unit of
reproduction. With the discovery and advancement of microscopes and magnification technology
over time, Robert Hooke was the first who studied the cork cells under the microscope and gave the
very significant theory on scientific study of cells, also known as ‘cell biology’ or ‘cell theory’. Cell
theory was eventually formulated in 1839. The three principles to the cell theory include that, all
living organisms are composed of one or more cells, the cell is the basic unit of structure and
organization in organisms and the cells arise from pre-existing cells. In this unit, will study about the
significance and characteristic features of a cell, cell as basic unit of life, various components of a cell,
and the structural organization of Prokaryotic and Eukaryotic cells

FUNDAMENTALS OF CELL THEORY :

By definition, a ‘cell’ is the fundamental and structural unit of all living organisms. It is the
smallest biological, structural and functional unit of all plants and animals. Therefore, cells are
called the ‘Building Blocks of Life’ or the ‘Basic units of Life’. Organisms made up of a single cell
are termed as ‘unicellular’ whereas organisms made up of many cells are termed as
‘multicellular’. Cells perform many different functions within a living organism, such as
digestion, respiration, reproduction, etc., and keep it alive. For example, within the human body
there are various types of cells which perform specific functions. Basically, the cells form tissues
and multiple tissues make up an organ, different organs create an organ system, such as
digestive system, respiratory system, circulatory system, nervous system, etc., to perform
specific functions in the human body and any other living organism. Therefore, Cells, Tissue,
Organ, System, Organism/Human Body Fundamentally, all organisms are composed of
structural and functional units of life called ‘cells. The body of some organisms like bacteria,
protozoans and some algae are made up of a single cell while the body of fungi, plants and
animals are composed of many cells. Human body is built of about one trillion cells. Cells vary in
size and structure as they are specialized to perform different functions. But the basic
components and functions of the cell are common to all cells.

Landmarks in Cell Study:

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Soon after Anton van Leeuwenhoek invented the microscope, Robert Hooke in 1665 observed
a piece of cork under the microscope and found it to be made of small compartments which he
called ‘cells’, in Latin ‘cell’ means ‘small room’. In 1672, Leeuwenhoek observed bacteria, sperm
and red blood corpuscles, all of which were cells. In 1831, Robert Brown, an Englishman
observed that all cells had a centrally positioned body which he termed the nucleus.

Anton van Leeuwenhoek is another scientist who saw these cells soon after Hooke. He made use of a
microscope containing improved lenses that could magnify objects almost 300-fold or 270x.
Leeuwenhoek studied the tiny organisms under the microscope and named them ‘animalcules’, which
included protozoa and other unicellular organisms, like bacteria. Antony van Leeuwenhoek is regarded
as the ‘Father of Microbiology’. He is known for the discovery of bacteria. In 1838, M.J. Schleiden and
Theodore Schwann formulated the ‘cell theory’. The cell theory formulates the following three
principles:

• All organisms are composed of cells.

• Cell is the structural and functional unit of life.
• Cells arise from pre-existing cells
SIZE AND STRUCTURE OF CELL

There are many cells in an individual, which performs several functions throughout the life. The
different types of cell include- prokaryotic cell, plant and animal cell. The size and the shape of
the cell range from millimeter to microns, which are generally based on the type of function that
it performs. A cell generally varies in their shapes. A few cells are in spherical, rod, flat, concave,
curved, rectangular, oval and etc. These cells can only be seen under microscope.

Different types of cells

different types of cells all referenced to a standard E. coli ruler of 1 micron

(A) The protist Giardia lamblia, (B) a plant cell, (C) a budding yeast cell, (D) a red blood cell, (E) a
fibroblast cell, (F) a eukaryotic nerve cell, and (G) a rod cell from the retina.
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Cell Size

One may wonder why all cells are so small. If being able to store nutrients, is beneficial to the
cell, how come there are no animals existing in nature with huge cells? Physical limitations
prevent this from occurring. A cell must be able to diffuse gases and nutrients in and out of the
cell. A cell's surface area does not increase as quickly as its volume, and as a result a large cell
may require more input of a substance or output of a substance than it is reasonably able to
perform. Worse, the distance between two points within the cell can be large enough that regions
of the cell would have trouble communicating, and it takes a relatively long time for substances
to travel across the cell.

That is not to say large cells don't exist. They are, once again, less efficient at exchanging
materials within themselves and with their environment, but they are still functional. These cells
typically have more than one copy of their genetic information, so they can manufacture proteins
locally within different parts of the cell. Features of such large cells are following:

 Is limited by need for regions of cell to communicate

 Diffuse oxygen and other gases
 Transport of mRNA and proteins
 Surface area to volume ratio limited

Larger cells typically:

 Have extra copies of genetic information

 Have slower communication between parts of cell

Various types of cells ranging in different sizes

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The shapes of cells are quite varied with some, such as neurons, being longer than they are wide
and others, such as parenchyma (a common type of plant cell) and erythrocytes (red blood cells)
being equidimensional. Some cells are encased in a rigid wall, which constrains their shape,
while others have a flexible cell membrane (and no rigid cell wall).

The size of cells is also related to their functions. Eggs (or to use the Latin word, ova) are very
large, often being the largest cells an organism produces. The large size of many eggs is related
to the process of development that occurs after the egg is fertilized, when the contents of the egg
(now termed a zygote) are used in a rapid series of cellular divisions, each requiring tremendous
amounts of energy that is available in the zygote cells. Later in life the energy must be acquired,
but at first a sort of inheritance/trust fund of energy is used. Cells range in size from small
bacteria to large, unfertilized eggs laid by birds and dinosaurs. Here are some measurements
and conversions that will aid your understanding of biology.

1 meter = 100 cm = 1,000 mm = 1,000,000 µm = 1,000,000,000 nm

1 centimeter (cm) = 1/100 meter = 10 mm

1 millimeter (mm) = 1/1000 meter = 1/10 cm

1 micrometer (µm) = 1/1,000,000 meter = 1/10,000 cm

1 nanometer (nm) = 1/1,000,000,000 meter = 1/10,000,000 cm

PROKARYOTIC AND EUKARYOTIC CELL

Body of all living organisms except virus has cellular organization and may contain one or many
cells. The organisms with only one cell in their body are known as unicellular (bacteria, protozoa
etc.) and organisms with many cells in their body are known as multicellular organisms (most
plants and animals). Any cellular organization may contain only one type of cell from the
following types:

A- Prokaryotic cell and B- Eukaryotic cell. These terms were suggested by Hans Ris in the 1960‘s.

PROKARYOTIC CELL

The prokaryotic (Gr., pro= primitive or before and karyon = nucleus) are small, simple, and most
primitive. They are probably first to come into existence perhaps 3.5 billion years ago. These
cells occur in bacteria (i.e., Mycoplasma, Cyanobacteria etc). Prokaryotic cell is a one envelope
system organized in depth. It consists of central nuclear components surrounded by cytoplasmic
ground substance, with whole enveloped by a plasma membrane. The cytoplasm of prokaryotic
cell lacks nuclear envelope and any other cytoplasmic membrane and well defined cytoplasmic
organelles.
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The size of prokaryotic cells ranges between 1 to 10 µm. They occur in a variety of forms.

Prokaryotic cell consists of three main components:

Outer covering:It is composed of inner cell or plasma membrane, middle cell wall and outer
slimy capsule.

Cell membrane:

Cell membrane made up of lipids and proteins, is thin and flexible and controls the movement of
molecules across the cell. Respiratory enzymes are carried by it for energy releasing reactions.
Mesosomes, the in-folds of plasma membrane bears respiratory enzymes and these are
considered analogous to mitochondria of eukaryotic cells. Similarly, the pigments and enzymes
molecules that absorb and convert the light into chemical energy in photosynthetic cells are also
associated with the plasma membrane’s in-folds called photosynthetic lamella. These lamellae
are analogous to the chloroplast of eukaryotic cells. Plasma membrane plays role in replication
and division of nuclear material. Since the in-folds remain continuous with the cell membrane,
they are not considered as separate compartments. Thus, prokaryotic cell is non-
compartmentalized.

Structure of a prokaryotic cell

Cell wall: It is a rigid or semi-rigid non-living structure that surrounds the cell membrane and
its thickness ranges between 1.5 to 100 µm. Chemically it is composedof peptidoglycans. Some
bacteria such as mycoplasmas lack cell wall.

Slimy capsule: A gelatinous coat outside the cell wall is the slimy capsule. It is composed of
largely of polysaccharides and sometimes it may have polypeptides and other compounds also.
It protects the cell against desiccation, virus attacks, phagocytosis and antibiotics
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Cytoplasm: Prokaryotic cytoplasm contains proteins, lipids, glycogen and inorganic ions along
with enzymes for biosynthetic reactions and ribosomes, tRNA and mRNA for protein synthesis.
Prokaryotic cytoplasm has some special features as follows:

 It lacks cell organelles like endoplasmic reticulum, mitochondria, Golgi apparatus,

Centrosomes, vacuoles, Lysosomes, microfilaments, intermediate filaments and
microtubules.
 The only cytoplasmic organelle found in prokaryotic cells is the ribosomes. They are
smaller than eukaryotic ribosomes i.e., 70S and lie free in the cytoplasm. They form poly-
ribosomes at the time of protein synthesis. They are the sites of protein synthesis.
 Like eukaryotic cells, the cytoplasm of prokaryotic cell does not show streaming
movement or cyclosis.
 Gas vacuoles are also formed in some prokaryotic cells.
 The cell does not show phagocytosis, pinocytosis and exocytose, substances enter and
leave the cell through the cell membrane.
 They may contain deposits of polysaccharides or inorganic phosphates.

Nucleoid: Nuclear envelope is absent in prokaryotic cell and the genetic material lies directly
into the cytoplasm. Such nuclear material is known as nucleoid. Nucleoid consists of greatly
coiled single pro-chromosome. It shows the following special features:

 A short and simple pro-chromosome is present which is attached at least at one point on
cell membrane.
 Mostly there is single copy of chromosome, the prokaryotic cell is haploid.
 The DNA is naked as it is not associated with basic histone proteins. It is double stranded,
helical and circular.
 The amount of DNA is lesser than eukaryotic cell and it codes fewer proteins. Replication
of DNA is continuous throughout the cell cycle. Transcription and translation occurs in
cytoplasm and processing of mRNA is not required.
 The processes like meiosis, gamete formation or fertilization are absent. Conjugation is
seen in some bacteria.
 Mitotic apparatus absent.
 There is no nucleolus.
 Cell membrane folds or mesosomes help to segregate the replicated products of
chromosomes into daughter cells.
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Plasmids: In some prokaryotic cells, in addition to nucleoid, a small circular double stranded
DNA molecule is present. It is called plasmid. Plasmids have 1000 to30,000 base pairs and they
generally encode proteins required by the organism to resist antibiotic and other toxic
material.

Flagellum: It is a whip like locomotory structure found in many bacteria. It is 150Å thick and
10 to 15µm long. As the flagellum does not have any surrounding membrane, it grows at the tip.
It has two main parts: Filament and basal body.

Filament- Filament extends out of cell into the medium and it is composed of many intertwined
spiral chains of the subunits of a protein called flagellin. Flagellin differs from actins or tubulin.

Basal Body- The basal body attaches the flagellum to the cell and generates the force to rotate
it. It is composed of many components and numerous proteins. It has two parts: shaft and hook.

Pili: These are short, rod like non-motile processes or fimbriae present on many bacteria. These
are formed of pilin protein. They are usually less than 10 nm thick. They help in attachment of
bacteria to surfaces or food or to one another. Tubular sex Pili are present in some bacteria.

Prokaryotic cells have all the biochemical mechanisms required to synthesize complex
organic materials from simple organic precursors necessary for life. Thus, inspite of being
simple in structure prokaryotes are more versatile in their syntheticactivities than eukaryotes.

Eukaryotic Cell

The Eukaryotic cells are essentially two envelope systems and they are very much larger than
prokaryotic cells. Secondary membranes envelop the nucleolus and other internal organelles
and to a great extent they pervade the Cytoplasm as the Endoplasmic reticulum. The Eukaryotic
cells are true cells which occur in the plants (from algae to angiosperms) and the animal (from
Protozoa to mammals). Though the Eukaryotic cells have different shape, size, and physiology;
all the cells are typically composed of plasma membrane, cytoplasm and its organelles, viz.
Mitochondria, Endoplasmic reticulum, ribosomes, Golgi apparatus etc; and a true nucleus. Here
the nuclear contents, such as DNA, RNA, Nucleoproteins and Nucleolus remain separated from
the Cytoplasm by the thin perforated nuclear membranes. Before going into the detail of cells
and its various components; it will be advisable to consider the general features of different
types of eukaryotic cells which are as follow:
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Structure of a Eukaryotic cell

Cell Shape: The basic shape of Eukaryotic Cells is Spherical; however the shape is ultimately
determined by the specific function of the cell. Thus, the shape of the cell may be Variable or
Fixed. Variable or irregular shape occurs in Amoeba and white blood cells or Leucocytes. Fixed
shape cells occur in almost all protests‘, plant, animals. In unicellular organisms the cells shape
is maintained by tough plasma membrane and exoskeleton. The shape of cells may vary from
animal to animal and from organ to organ. Even cells of the same organ may display variations
in the shape. Thus cells may have diverse shape such as Polyhedral, Flattened, Cuboidal,
Columnar, Discoidal, Spherical, Spindle Shaped, Elongated or Branched.

Cell Size: The eukaryotic cells are typically larger (mostly ranging between 10 to 100 µm) than
the prokaryotic cells (mostly ranging between 1 to 10 µm). Size of the cell of the unicellular
organisms is larger than typical multicellular organisms‘ cells. For example, Amoeba proteus is
biggest among the unicellular organisms. One species of Euglena is found up to 500 µm in length.
Diatoms have length of 200µm or more. The size of multicellular organism ranges between 20
to 30µm. Among animal‘s the smallest cells have a diameter 4µm. (e.g. Polocytes); Human
erythrocytes being 7 to 8 µm in diameter. Largest animal cell is the egg of Ostrich, having a
diameter of 18cm and the longest cell is human nerve cell, a meter long.

Cell Volume: The volume of cell is fairly constant for a particular cell type and is independent of
the size of the organism (Law of Constant Volume). For example, kidney or liver cells are about
the same size in the bull horse and mouse. The difference in the total mass of organ depend upon
the number not on the volume of the cells. If a cell is to be efficient, the ratio of volume to surface
should be within a limited range. An increase in the cell volume is accompanied by much smaller
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expansion in surface area of the cells. In other words, a large cell has a proportionately smaller
surface area and a higher volume: Surface ratio than a small a cell.

Cell Number: The number of cell present in organism is varies from a single cell in unicellular
organism to many cell in multi cellular organism. The number of cell in multicellular organism
usually remains correlated with the size of organism and, therefore, a small sized organism has
a less number of cells in comparison to large sized organism. Further, the number of cells in most
of multicellular organism is indefinite, but the number of cells may be fixed in some multicellular
organism. For example, in rotifers, numbers of nuclei in the various organs are found to be
constant in any given species. The phenomenon of cells or nuclear constancy is called Eutely. In
one species of rotifers, Martini (1912) always found 183 nuclei in the brain, 39 in stomach and
so on.

Structure:

Cell Wall: The outermost structure of most plant cells is a dead and rigid layer called cell wall.
It is mainly composed of carbohydrates such as cellulose, pectin hemicelluloses and lignin and
certain fatty substances like waxes. There is pectin- rich cementing substance between the walls
of adjacent cells which is called middle lamella. The cell wall which is formed immediately after
the division of cell, constitute the primary cell wall. In certain types of cells such as phloem and
xylem, an additional layer is added to the inner surface of primary cell wall called, secondary cell
wall and it consist mainly of cellulose, hemicelluloses and lignin. In many plant cells, there are
tunnels running through the cell wall called Plasmodesmata which allow communication with
the other cells in a tissue.

Plasma Membrane: Every kind of animal cell is bounded by a living, extremely thin and delicate
membrane called Plasma lemma, cell membrane or plasma membrane. In plant calls plasma
membrane occurs just inner to cell wall, bounding the cytoplasm. The plasma membrane
exhibits a tri- laminar structure with a translucent layer sandwiched between two dark layers.
The plasma membrane is selectively permeable membrane; its main function is to control
selectively the entrance and exit of materials. This allows then cell to maintain a constant internal
environment (Homeostasis). Molecules of water, oxygen, carbon-dioxide, glucose etc., are
transported across the plasma membrane takes place by various means such as osmosis,
diffusion, and active transportation.

Cytosol: The plasma membrane is followed by the colloidal organic fluid called matrix or cytosol.
The cytosol is aqueous part of cytoplasm and nucleoplasm. Cytosol is particularly rich in
differentiation cells and many fundamental properties of cells are because of this part of
cytoplasm. The cytosol serves to dissolve or suspend the great verity of small molecules
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concerned with cellular metabolism, e.g., glucose amino acids, nucleotides, vitamins, minerals,
oxygen. In all type of cells, cytosol contains the soluble proteins and enzymes which form 20-
25% of the total protein content of the cells. In many types of cells, the cytosol is differentiated
into two parts:

 Ectoplasm or cell cortex is the peripheral layer of cytosol which is relatively non granular,
viscous, clear and rigid.
 Endoplasm is the inner portion of cytosol which is granular and less viscous.

Differences between Prokaryotic Cells and Eukaryotic Cells

The internal organization of eukaryotic cell is more developed than prokaryotic cellsfrom
which they are believed to have been evolved.

Prokaryotic Cells Eukaryotic Cells

1. A prokaryotic cell is surrounded by a A eukaryotic cell issurrounded by a double

single membrane layer. membranelayer.

2. In most cases the cell wall surrounds the Cell wall is present in protists, most fungi
plasmamembrane and it is composed of and plants and is composed of chitin in
carbohydrates, lipids proteins and most fungi and or cellulose in others.
certain amino acids

3. Respiratory enzymes are present on cell Absent on the cellmembrane

membranes.

4. Thalakoids occurs free incytoplasm. They occur within thechloroplast.

5. Cytoplasm lacks organelles like All the cell organelles are present in the cell
centrosomes, endoplasmic reticulum, along with ribosomes.
mitochondria, Golgi apparatus,
microfilaments, intermediate filaments,
microtubules and micro bodies. While
ribosomesare present

6. Gas vacuoles may occur while sap Sap vacuoles are commonly present.
vacuoles are absent.

7. 70S ribosomes are present that lie free in 80S ribosome’s arepresent, either free or
cytoplasm orattached to mRNA. bound to ER and nuclear envelope or
mRNA.

8. Endocytosis and exocytosedo not occur. These processes take place in many
protists and in animals.

9. Process of meiosis or gamete formation In these cells the process of meiosis,

or true fertilization does not occur. gamete formation and true fertilization
occur in most cases of sexualreproduction.
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10. Cells are haploid. Cells are diploid, while haploid cells also
occur.

11. Most prokaryotes are asexual organisms. Most of them are sexual organisms.

12. Nuclear envelope is absent and nuclear Nuclear envelope surrounds the nuclear
material lie in cytoplasm and is called material. The structure is called nucleus. It
nucleoid. Nucleoid contains a single contains two to manychromosomes.
chromosome.

13. Nucleolus absent. One or more nucleoliare present within the

nucleus.

14. Circular DNA is present without Nuclear DNA is linear and is associated
associatedproteins. with proteins, while extranuclear DNA is
present without proteins.

15. Flagella if present aresimple, consist of a Flagella, if present are complex, have 9+2
single fibril and are formed of a protein pattern of microtubulesformed of a protein
flagellin. tubulin.

16. Plasmids and pili occur in many These stuctures are absent.
prokaryotic cells.

Cell wall

The cells of majority of organisms are enclosed in a protective cell wall surrounding a fragile
plasma membrane. It determines cell shape and prevents cells from shrinking and bursting due
to changes in osmotic pressure. Despite a common role the cell wall of bacteria and eukaryotes
are structurally divergent. This section deals with some of the variations in cell wall composition
encountered in nature.

Prokaryotic Cell wall

The differential staining technique developed by Christian Gram in 1884 broadly divided bacteria
into Gram positive and Gram-negative organisms. We still do not have a complete understanding
of the basis of Gram reaction although differences in cell wall structure are largely responsible
for it. We begin this subsection by highlighting the major structural differences between them.

Cell wall gram positive bacteria

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In Gram positive bacteria such as species of Bacillus the plasma membrane is surrounded by a
thick cell wall made up of several sheets of peptidoglycan or murein (20-80nm) stacked one
upon another. Additionally, many bacteria have acidic teichoic acid polymers embedded in the
cell wall. The peptidoglycan is a branched polymer of linear glycan chains made up of a repeating
disaccharide, namely N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM).

The two sugars are linked via a β (1 4) glycosidic linkage. Each NAM residue is linked to a tetra-
or penta- peptide that generally has few D-amino acids. The peptidoglycan is cross linked either
directly with the free carboxyl and amino groups of tetra peptides on different chains or via a
penta glycine linker. The resultant multilayered covalently cross linked structure is much
stronger than the cell wall of Gram negative bacteria. Interestingly, the unique structure of their
cell walls also makes them vulnerable to some antibiotics. The teichoic acid polymers are
negatively charged phosphorylated sugar alcohols. The repeating units (10-50) are linked via
phosphodiester bonds. Depending upon the organism the building block may vary. The 1, 3 poly
(glycerol phosphate) teichoic acid are present in many species of bacteria and 1, 5 poly (ribitol
phosphate) teichoic acid occur in many lactobacilli and Staphylococci. These polymers may have
D- amino acids or other sugars as substituents. Teichoic acids are attached covalently to murein
through a linkage unit.

Cell wall Gram negative bacteria

The cell wall of Gram negative bacteria is more complex than Gram positive bacteria. It has a dual
membrane system consisting of an inner peptidoglycan and a permeable outer membrane, LPS
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The structure of peptidoglycan is similar to that of the Gram positive bacteria except that it is
much thinner (2-7nm) and represents 5-10% of the wall weight. The lipopolysaccharide (LPS)
has phospholipids, protein and lipopolysaccharides. The structure of LPS can be subdivided into
three parts – O-antigen, core polysaccharide and lipid A. The O-antigen is a repeating chain of
tetra or pentasaccharides and shows enormous variability. It accounts for type specific
immunological reactions. The core polysaccharide of nearly all LPS has D-glucose, D-galactose, N-
acetyl glucosamine, a heptose and an unusual octose derivative 2-keto 3-deoxyoctonate (KDO).
Finally, lipid A is the least variable part of LPS and is responsible for all the ills and benefits that
endotoxins (an earlier name of LPS) inflict on the host. It has two N-acetyl glucosamine (NAG)
phosphates or pyrophosphate residues to which saturated fatty acids are attached. Lipid A is
linked to KDO residue of the core polysaccharide. The outer membrane also has Braun’s
lipoprotein that is covalently linked to peptidoglycan. The aqueous channels (porins) interrupt
the membrane and facilitate movement of low molecular weight hydrophilic molecules.

Eukaryotic Cell Wall

The cell wall of eukaryotes (fungi, algae, and higher plants) is composed principally of
polysaccharides. In this section we will study two major structural variants seen in eukaryotes –
cellulose based or largely non cellulose based.

Cell wall of Fungi

The cell wall composition is variable among different groups of fungi and our present knowledge
is incomplete. It is recognised that true fungi have chitin and glucans in their cell wall and majority
of fungi lack cellulose (Fig 6.6a). Chitin is a linear polymer of NAG residues (Fig.6.6b). In some
zygomycetes fungi chitin is partially deacetylated by chitin deacetylase enzyme and so the cell
wall contains chitosan (deacetylated chitin). Both chitin and its variant chitosan form microfibrils
like cellulose and associates with non fibrillar material. The other polysaccharides include mainly
β-glucans that are generally β (1, 3) or β (1, 6) linked. The majority of glycoproteins are mannans
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or mannoproteins. Some proteins are also anchored to the cell membrane that helps in
maintaining cell wall integrity. The cell wall of yeast is composed of microfibrils of mannans and
β-glucans.

Cell wall of Algae

The cell walls of most algae and higher plants are composed of cellulose which is a linear
homopolymer of glucose residues linked by β (1 4) linkage (Fig.6.7a). It is the most abundant
organic substance found in nature. The most stable conformation of cellulose fibrils is an
extended straight chain structure in which each monomer is turned 180o relative to the
preceding one. In higher plants 30-36 chains join in the same polarity to form a single microfibril,
stabilized by hydrogen bonding. The microfibrils assemble to form macrofibrils

Plant

Plant cell walls also consist of a complex mixture of polysaccharides that provide a gel like
medium in which cellulose microfibrils are embedded. The matrix polysaccharides are branched
heteropolysaccharides; earlier divided into pectins and hemicellulose on the basis of their
solubility. Our present knowledge of the chemical complexity of these polymers has led to a
nomenclature based on their structure. The pectins are acidic polysaccharides with an abundance
of D-galacturonic acid residues such as homogalacturonans. The hemicelluloses are represented
by xyloglucans that may be heteroxylans (arabinoxyloglucans) and heteromannans
(galactomannans).
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Despite the apparent diversity, cell walls are classified into primary and secondary walls. Primary
walls are formed by growing cells and are relatively unspecialized and similar in molecular
architecture in all cell types. They are composed predominantly of polysaccharides together with
lesser amount of structural glycoproteins (extensins), phenolic esters, minerals and enzymes. The
major polysaccharides in the primary wall are cellulose, hemicellulose and pectin in almost equal
proportions (Fig. 6.8) and cellulose fibers are randomly arranged.

The secondary cell wall is located between the primary cell wall and plasma membrane (Fig. 6.9).
A cell starts producing secondary cell wall after primary wall is complete and cell growth
(enlargement) has ceased. The secondary cell wall has a higher density of cellulose microfibrils
(50-80%), along with other polysaccharides, lignin, and glycoproteins. Lignin largely replaces
xyloglucans, glucoarabinoxylans and pectins in the matrix and it is cross linked to microfibrils
that is largely responsible for much of its strength and density of wood. The secondary walls are
frequently laid down in layers in which cellulose fibers differ in orientation, forming a laminated
structure that greatly increases cell wall strength. It is more rigid, and less permeable to water
than the primary cell wall. It is also more resistant to degradation.

The Structure of Primary Cell Wall

Model of the Secondary Cell Wall

Extracellular Matrix (ECM)

In the absence of cell wall many animal cells / tissues are surrounded by an ECM or ground
substance — an organized network of extracellular materials present beyond the immediate
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vicinity of the plasma membrane. Most cells in multicellular organisms secrete proteins and
heteropolysaccharides into the extracellular space where they self assemble to form an organised
meshwork characteristic of the ECM. The ECM is more than inert nonspecific glue that holds cells
together; it often plays a key regulatory role in determining shape and activities of the cell.

One of the best studied extracellular matrices is the basement membrane (or basal lamina). It is
a continuous sheet that surrounds nerve fibers, muscles, and fat cells and underlies the basal
surface of epithelial tissues (epidermis of the skin), the lining of digestive and respiratory tracts
and endothelial lining of blood vessels. Basement membranes provide mechanical support for
attached cells, generate signals that dictate cell survival, serve as a substratum for cell migration,
separate adjacent tissues within an organ, and act as a barrier to the passage of macromolecules.
However, the ECM is most abundant in connective tissues. For example, the loose connective
tissue beneath epithelial cell layers consists predominantly of an ECM in which fibroblasts are
distributed. The ECM is mainly composed of tough fibrous proteins embedded in a gel-like
polysaccharide ground substance - a design that is basically similar to that of plant cell walls. It
also has adhesion proteins that link components of the matrix to one another and to attached
cells.

An Overview of the Organization of the ECM.

Matrix Portion

Unlike most proteins that are compact and globular in shape, those of the extracellular space are
fibrous with extended conformation. In the reticular ECM surrounding the fibroblasts and other
connective tissue cells the fibrous proteins include fibrillar collagen, elastin and fibronectin
whereas basement membrane ECM underlying the epithelial cells has specialised collagens and
laminin. The major structural protein of ECM is Collagen.
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The collagens are a large family of glycoproteins containing at least 27 different members which
are produced by fibroblasts, osteoblasts, chondrocytes and epithelial cells. Each collagen type is
restricted to particular locations within the body, but two or more different types are often
present together in the same ECM. Additional functional complexity is provided by mixing several
collagen types within the same fiber.

The most abundant collagen is type I collagen. It is one of the fibril forming collagens. Their
polypeptide chains consist of approximately 1000 amino acids or 330 Gly-X-Pro or Gly-X-4-Hyp
repeats. After secretion as soluble precursors (procollagens) they are cleaved and assembled into
collagen fibrils in which triple helical molecules are associated in regular staggered arrays.

All members of the collagen family share at least two important structural features. First, three
separate polypeptides, called α-chains are supertwisted about each other. The superhelical
twisting is right handed in collagen (Fig.6.11a and b). Second, many of the proline (and lysine)
residues of each α -chain are hydroxylated. The tight wrapping of α –chains in the triple helix
provides enormous tensile strength that can vary depending on the way they associate to form
these supra molecular assemblies.

In addition to collagen, connective tissue ECM has elastin and fibronectin. The former is present
as bundles and provides elasticity to tissues and organs like lungs and arteries while the latter
binds integrins and other ECM components such as collagen or proteoglycans and have a role in
cell adhesion. The major ECM protein of the basal lamina is laminin. It is a heterotrimeric
glycoprotein that can bind collagen, integrins and other cellular proteins. It can also bind itself to
form sheets in the basal lamina and has a role in cell adhesion.

Cell-Matrix Interactions

The structural components of the ECM such as proteoglycans bind to cell surface receptors and
influence their ability to convey signals. The most important family of cell surface receptors that
integrate extracellular and intracellular environments belongs to the integrin family. They are
heterodimers of noncovalently linked α- and β- chains. About two dozen different integrins have
been identified, each with a specific distribution. It was in recognition of their role in maintaining
 18

structural integrity of cells and tissues they were named ‘integrins’. They bind to various ligands
that are present in the extracellular environment and transduce signals by interacting either
directly or indirectly with other proteins to influence events within the cell. Some integrins like
LFA-1 (integrin) on one cell interact with LFA-3 (an ICAM) on another cell to mediate cell-cell
interactions.

Integrins have been implicated in two major types of activities. First they hold cells (cellular
“glue”) in place by attaching at one end to the ECM components / to complementary molecules
on other cells and at the other end to the cytoskeleton. Second they mediate “outside-in” signaling
by relaying signals from matrix (or other cells) to the cell interior. In response to signals the cells
respond in different ways such as changing shape, migration, proliferation, and differentiation.

The binding of integrin to ligands such as fibronectin or collagen induces a conformational change
at the cytoplasmic end of the integrin. This in turn alters the way the integrin interacts with
cytoplasmic proteins like kinases. These kinases can then phosphorylate other proteins, initiating
a chain reaction.

A Focal Adhesion Showing the Concentration of Integrins

Integrins respond to messages received from inside the cell as well (insideout-signaling). They
can change their ligands or the strength with which they bind to them in response to signals.
Inside-out-signaling is best documented in case of blood platelets. They circulate in blood singly
and are non adherent. When the endothelial linings of blood vessels are injured, blood platelets
stick to the ECM (without the help of integrins) in the exposed areas. This attachment (or the
binding of thrombin) signals the cytoplasm that results in an inside-out activation of the integrins
on the platelet surface. In this case integrins become more adhesive and better in picking up
circulating molecules of fibrinogen that forms molecular bridges with more platelets and to the
 19

matrix. The large aggregates of platelets and proteins make a dense meshwork of cells and fibers
(clots), preventing blood loss

Evolution of cell

All the living organisms are made up of cells. A living cell may be defined as a compartment
enclosed by a membrane that consists of several chemical constituents and is capable of
propagating itself by division. Activity of an organism is dependent on the activities of its cells,
individually and collectively. Basic structures and functions are common in all cells with certain
differences according to their specific needs. As you have already studied in Unit 12 of the
Foundation Course in Science and Technology, there are two stages in evolution of the cell;
chemical evolution and biological evolution. Chemical evolution started with the formation of
organic molecules on the earth which had no life until then. We call it prebiotic earth. Some of
these early molecules developed the ability of producing an exact copy of their ownself by self-
replication. We can mark this event as the origin of life. Later on, during the course of biological
evolution, molecular aggregates organised and formed the cell which was different from the cell
as it exists today. We shall trace these events which are based partly on speculation and partly
on experimental evidence.

The origin of a true life from, eubiont from a protobiont involves the acquisition of three major
features:

1) assembly of phospholipids and proteins into a cell membrane forms the boundary of the cell,

2) development of protein-directed metabolic pathways to utilise the organic molecules for

energy needs, and

3) formation of nucleic acid molecules that are not only capable of self-replication but are also
capable of storing genetic information for the synthesis of proteins.

Since we have no definite idea of the smcture of a protobiont, it is difficult to speculate the manner
in which a eubiont evolved from a protobiont. All the same, it is certain that a eubiont comparable
to the simplest living cell today must have originated first

It is quite likely that the formation of a boundary membrane would have taken place before the
acquisition of a genetic information system and metabolic pathways. A separation of the cell
interior from the outside, would permit sufficient interaction of molecules inside the cell.
 20

Prebiotic Molecules, Ammonia, Methane, Water, Formaldehyde etc

Amino acids, nucledtides, sugars

Proteins. Nucleic acid, lipids, polysaccharides, ATP

Metabolic pathways Plasma Membrane Genetic Information System

First procaryote 3.5 and 3.0 x l09 years ago

First Eucaryote 0.9 x l09 years ago

First Multicellular Organisms

Procaryotes to Eukaryotes

Procaryotes are the simple, unicellular organisms which do not have well defined nucleus,
whereas eucaryotes are the complex; multicellular organisms with well defined nucleus.
Procaryotes were originated about three and a half billion years ago, whereas eucaryotes
originated about one and a half billion years ago. Thus, it appears that eucaryotes evolved later
than the procaryotes. But in biochemical details the procaryotes and eucaryotes are very similar.
Therefore, we can say that they have a common origin.

Eucaryotes, like procaryotes. have the same biochemical pathways for respiration both
anaerobic and aerobic and for photosynthesis. The eucaryotes not only attained larger size and
complex structure but also developed into multicellular organisms. Earlier each form of cell
which was thought as an autonomous arid self-sufficient unit, gradually became dependent on
each other in larger organism is leading to division of labour within organisms. This type of
cooperation between cells eventually led to the development of higher forms of life as they exist
today.

Virus

Structure of Viruses

A fully assembled infectious virus is called as a “VIRION” (the intact virus unit). The main
function of virion is to deliver its DNA or RNA genome into the host cell. Each viral species has a
very limited host range. The term ‘virus’ and ‘virion’ bear the same connotation and are often
interchangeable
 21

Each virion is composed of two or three parts: (i) the genetic material made from either DNA
or RNA, (ii) a protein coat, called the capsid, (iii) an envelope of lipid (Figure 1). A protein coat
functions as a shell to protect the viral genome from nucleases. The subunit of capsid is called
as ‘capsomere’. The nucleic acid together with the capsid is knownas nucleocapsid. Some
viruses have membranous envelope that lies outside the nucleocapsid, and are referred as
enveloped viruses, while one lacking them are called as naked viruses. In the enveloped viruses,

nucleocapsid is surrounded by a lipid bilayer and glycoprotein. Enveloped viruses often

exhibit a fringe of glycoprotein spikes called as peplomers

Viruses exhibit different shapes and symmetry (Table 1). The symmetry refers to the way in
which the capsomeres are arranged in the virus capsid. Accordingly following are the four
categories

Shapes Polyhedral Helical viruses Complex Enveloped

of viruses viruses viruses
Virues They are alsocalled The nucleic acid These viruses are These virusesare
icosahedral viruses genome in these composed of surrounded by a
because of their viruses, is wound various proteins membrane made
symmetry. These inside acylindrical that functions up of glycoproteins
viruses arecomposed protein capsid with to protect the that seek out cells
of polyhedral protein Helical symmetry. genome, attach to to infect. E.g.:
shells. cells, and Influenza and HIV
E.g.: TMV and
introduce the
E.g.: Poliovirus, M13
nucleic acid
herpes simplexvirus
inside E.g.:Vacinia
virus
 22

Structure and Function

Viruses are small obligate intracellular parasites, which by definition contain either a RNA or
DNA genome surrounded by a protective, virus-coded protein coat. Viruses may be viewed as
mobile genetic elements, most probably of cellular origin and characterized by a long co-
evolution of virus and host. For propagation viruses depend on specialized host cells supplying
the complex metabolic and biosynthetic machinery of eukaryotic or prokaryotic cells. A
complete virus particle is called a virion. The main function of the virion is to deliver its DNA or
RNA genome into the host cell so that the genome can be expressed (transcribed and translated)
by the host cell. The viral genome, often with associated basic proteins, is packaged inside a
symmetric protein capsid. The nucleic acid-associated protein, called nucleoprotein, together
with the genome, forms the nucleocapsid. In enveloped viruses, the nucleocapsid is surrounded
by a lipid bilayer derived from the modified host cell membrane and studded with an outer layer
of virus envelope glycoproteins.

Classification of Viruses

Morphology: Viruses are grouped on the basis of size and shape, chemical composition and
structure of the genome, and mode of replication. Helical morphology is seen in nucleocapsids of
many filamentous and pleomorphic viruses. Helical nucleocapsids consist of a helical array of
capsid proteins (protomers) wrapped around a helical filament of nucleic acid. Icosahedral
morphology is characteristic of the nucleocapsids of many “spherical” viruses. The number and
arrangement of the capsomeres (morphologic subunits of the icosahedron) are useful in
identification and classification. Many viruses also have an outer envelope.
 23

Chemical Composition and Mode of Replication: The genome of a virus may consist of DNA or
RNA, which may be single stranded (ss) or double stranded (ds), linear or circular. The entire
genome may occupy either one nucleic acid molecule (monopartite genome) or several nucleic
acid segments (multipartite genome). The different types of genome necessitate different
replication strategies.

Retro Viruses

 Retro viruses are single stranded RNA containing animal viruses.

 Retroviruses are named for an enzyme known as reverse transcriptase, which were
discovered by Howard Temin and David Baltimore independently.
 These replicate through DNA intermediates, via a process of reverse transcription, in
presence of reverse transcriptase enzyme.
 Retroviruses cause tumour growth and certain cancers in animals and are also
associated with slow infections.
 HIV (Human Immunodeficiency Virus) is a famous example of retro virus causing AIDS
(acquired Immune Deficiency Syndrome).

Tobacco Mosaic Virus (TMV)

 W.M.Stanley in 1935 first time isolated TMV in its crystalline formand was awarded
Noble prize. This was the first isolation of a virus.
 TMV is a simple rod shaped helical virus (Figure 3).
 It consists of a single stranded RNA (5.6%) enveloped by a proteincoat (94.4%)
 R.Franklin calculated 2130 capsomeres in a complete helical rod ofTMV.
 TMV penetrates and enter the host cell in toto
 24

Bacteriophages

 Bacteriophages are the viruses which infect bacteria. They arecommonly called as
"phages" or "coliphages"
 The body of the typical bacteriophage (T-phage) consists of head andtail. Head being
hexagonal in appearance and the tail cylindrical.
 Bacteriophage resembles with living organisms in having DNA as thegenome.
 The proteinaceous body of the bacteriophage remains outside and thenucleic acid
enters the host cell at the time of infection.
 Lambda phage is also a type of bacteriophage and its name was coinedby A.Lwoff.
 The credit for making this discovery goes to E.Twort and de.Herelle

Structure and Function

Viruses are inert outside the host cell. Small viruses, e.g., polio and tobacco mosaic virus, can even
be crystallized. Viruses are unable to generate energy. As obligate intracellular parasites, during
replication, they fully depend on the complicated biochemical machinery of eukaryotic or
prokaryotic cells. The main purpose of a virus is to deliver its genome into the host cell to allow
its expression (transcription and translation) by the host cell.
 25

A fully assembled infectious virus is called a virion. The simplest virions consist of two basic
components: nucleic acid (single- or double-stranded RNA or DNA) and a protein coat, the capsid,
which functions as a shell to protect the viral genome from nucleases and which during infection
attaches the virion to specific receptors exposed on the prospective host cell. Capsid proteins are
coded for by the virus genome. Because of its limited size the genome codes for only a few
structural proteins (besides non-structural regulatory proteins involved in virus replication).
Capsids are formed as single or double protein shells and consist of only one or a few structural
protein species. Therefore, multiple protein copies must self assemble to form the continuous
three-dimensional capsid structure. Self assembly of virus capsids follows two basic patterns:
helical symmetry, in which the protein subunits and the nucleic acid are arranged in a helix, and
icosahedral symmetry, in which the protein subunits assemble into a symmetric shell that covers
the nucleic acid-containing core.

Classification of Viruses

Viruses are classified on the basis of morphology, chemical composition, and mode of replication.
The viruses that infect humans are currently grouped into 21 families, reflecting only a small part
of the spectrum of the multitude of different viruses whose host ranges extend from vertebrates
to protozoa and from plants and fungi to bacteria.

Morphology

Helical Symmetry

In the replication of viruses with helical symmetry, identical protein subunits (protomers) self-
assemble into a helical array surrounding the nucleic acid, which follows a similar spiral path.
Such nucleocapsids form rigid, highly elongated rods or flexible filaments; in either case, details
of the capsid structure are often discernible by electron microscopy. In addition to classification
as flexible or rigid and as naked or enveloped, helical nucleocapsids are characterized by length,
width, pitch of the helix, and number of protomers per helical turn. The most extensively studied
helical virus is tobacco mosaic virus. Many important structural features of this plant virus have
been detected by x-ray diffraction studies.
 26

Icosahedral Symmetry

An icosahedron is a polyhedron having 20 equilateral triangular faces and 12 vertices (Fig. 41-3).
Lines through opposite vertices define axes of fivefold rotational symmetry: all structural
features of the polyhedron repeat five times within each 360° of rotation about any of the fivefold
axes. Lines through the centers of opposite triangular faces form axes of threefold rotational
symmetry; twofold rotational symmetry axes are formed by lines through midpoints of opposite
edges. An icosaheron (polyhedral or spherical) with fivefold, threefold, and twofold axes of
rotational symmetry

Viruses were first found to have 532 symmetry by x-ray diffraction studies and subsequently by
electron microscopy with negative-staining techniques. In most icosahedral viruses, the
protomers, i.e. the structural polypeptide chains, are arranged in oligomeric clusters called
capsomeres, which are readily delineated by negative staining electron microscopy and form the
closed capsid shell. The arrangement of capsomeres into an icosahedral shell permits the
classification of such viruses by capsomere number and pattern. This requires the identification
of the nearest pair of vertex capsomeres (called penton: those through which the fivefold
symmetry axes pass) and the distribution of capsomeres between them

Virus Core Structure

Except in helical nucleocapsids, little is known about the packaging or organization of the viral
genome within the core. Small virions are simple nucleocapsids containing 1 to 2 protein species.
The larger viruses contain in a core the nucleic acid genome complexed with basic protein(s) and
protected by a single- or double layered capsid (consisting of more than one species of protein)
or by an envelope

Chemical Composition and Mode of Replication

RNA Virus Genomes

 27

RNA viruses, comprising 70% of all viruses, vary remarkably in genome structure. Because of the
error rate of the enzymes involved in RNA replication, these viruses usually show much higher
mutation rates than do the DNA viruses. Mutation rates of 10-4 lead to the continuous generation
of virus variants which show great adaptability to new hosts. The viral RNA may be single-
stranded (ss) or double-stranded (ds), and the genome may occupy a single RNA segment or be
distributed on two or more separate segments (segmented genomes). In addition, the RNA strand
of a single-stranded genome may be either a sense strand (plus strand), which can function as
messenger RNA (mRNA), or an antisense strand (minus strand), which is complementary to the
sense strand and cannot function as mRNA protein translation. Sense viral RNA alone can
replicate if injected into cells, since it can function as mRNA and initiate translation of virus-
encoded proteins. Antisense RNA, on the other hand, has no translational function and cannot per
se produce viral components.

DsRNA viruses, e.g., members of the reovirus family, contain 10, 11 or 12 separate genome
segments coding for 3 enzymes involved in RNA replication, 3 major capsid proteins and a
number of smaller structural proteins. Each segment consists of a complementary sense and
antisense strand that is hydrogen bonded into a linear ds molecule. The replication of these
viruses is complex; only the sense RNA strands are released from the infecting virion to initiate
replication.

The retrovirus genome comprises two identical, plus-sense ssRNA molecules, each monomer 7–
11 kb in size, that are noncovalently linked over a short terminal region. Retroviruses contain 2
envelope proteins encoded by the env-gene, 4–6 nonglycosylated core proteins and 3 non-
structural functional proteins specified by the gag-gene. The RT transcribes the viral ssRNA into
double-stranded, circular proviral DNA. This DNA, mediated by the viral integrase, becomes
covalently bonded into the DNA of the host cell to make possible the subsequent transcription of
the sense strands that eventually give rise to retrovirus progeny. After assembly and budding,
retroviruses show structural and functional maturation. In immature virions the structural
proteins of the core are present as a large precursor protein shell. After proteolytic processing by
the viral protease the proteins of the mature virion are rearranged and form the dense isometric
or cone-shaped core typical of the mature virion, and the particle becomes infectious.

DNA Virus Genomes

Most DNA viruses contain a single genome of linear dsDNA. The papovaviruses, comprising the
polyoma- and papillomaviruses, however, have circular DNA genomes, about 5.1 and 7.8 kb pairs
in size. DsDNA serves as a template both for mRNA and for self-transcription. Three or 2
 28

structural proteins make up the papovavirus capsid: in addition, 5-6 nonstructural proteins are
encoded that are functional in virus transcription, DNA replication and cell transformation.

Single-stranded linear DNA, 4–6 kb in size, is found with the members of the Parvovirus family
that comprises the parvo-, the erythro- and the dependoviruses. The virion contains 2–4
structural protein species which are differently derived from the same gene product. The adeno-
associated virus (AAV, a dependovirus) is incapable of producing progeny virions except in the
presence of helper viruses (adenovirus or herpesvirus). It is therefore said to be replication
defective.

Circular single-stranded DNA of only 1.7 to 2.3 kb is found in members of the Circovirus family
which comprise the smallest autonomously propagated viruses. The isometric capsid measures
17 nm and is composed of 2 protein species only.

Bacteria

The bacteria are amongst smallest organisms, most primitive, prokaryotic and microscopic
organisms. They occur almost everywhere; in air, water, soil and inside other organisms. They
lead either autotrophic or heterotrophic mode of existence.

Size of Bacteria: It ranges between 1-3µm and are barely visible under light microscope.

Shape of Bacteria: The three basic bacterial shapes are:

 Cocci – (spherical shaped). e.g., Diplococcus pneumonia, Streptococcus pyogenes etc.

 Bacilli – (rod-shaped) e.g. Mycobacterium, Clostridium botylinum etc.
 Spirilla (spiral or twisted) e.g. Treponema pallidum etc However pleomorphic bacteria can
assume several shapes.

Structure of Bacteria:

1. Plasma membrane- It is an ultra thin membrane 6-8 nm thick, chemically comprised of

molecules of lipids and proteins, arranged in a fluid mosaic pattern. Infoldings in it gives rise to
two main types of structures:
2. Mesosomes- (Also known as Chondriods); are extensions involving complex whorls of
convoluted membranes. They increase surface area of plasma membrane and enzymatic
contents.
3. Chromatophores- These are photosynthetic pigment- bearing membranous structures of
photosynthetic bacteria and are present as vesicles, thylakoids, tubes etc.
4. Cell Wall- It is strong and rigid and covers plasma membrane to provide chemical protection
and characteristic shape of bacteria. It is made up of peptidoglycan and contains muramic acid.
 29

5. Capsule- In some bacteria, cell wall is surrounded by an additional slime or gel layer called
capsule that acts as protective layer against viruses and phagocytes.
6. Cytoplasm- It is the ground substance surrounded by plasma membrane and is site of all
metabolic activities of bacteria. It consists of water, proteins, enzymes, different types of RNA
molecules and reserve materials like glucogen, volutin and sulphur. The dense nuclear areas of
cytoplasm contain 70S ribosomes granules, composed of RNA and protein and are the site of
protein synthesis.
7. Nucleoids- The nuclear membrane includes a single, circular and double stranded DNA
molecule often called as bacterial chromosome. It is not separated by nuclear membrane and is
usually concentrated in a specific clear region of the cytoplasm called nucleoid. It has no
ribosomes, nucleolus and histone proteins.
8. Plasmids – Many species of bacteria may also carry extrachromosomal genetic elements in the
form of small, circular, and closed DNA molecules called plasmids. They produce antibiotically
active protein or colicins which inhibit the growth of other bacterial strain in their vicinity. They
may also act as sex or fertility factors (F factor) which stimulate bacterial conjugation. R factors
are also plasmid carrying genes for resistance to drugs.
9. Flagella- Many bacteria are motile and contain one or more flagella for cellular locomotion. They
are 15-20nm in diameter and up to 20µm long. e.g., E.coli etc
10. Nutrition: They show diversity in their nutrition from being chemosynthetic, to photosynthetic;
but most of them are heterotrophic. Heterotrophic bacteria are mostly either saprophytic or
parasitic. Parasitic lives on the bodies of other organisms. Most bacteria are pathogenic.
11. Mode of Respiration: It is of both types; aerobic (which respire in the presence of oxygen.eg
Lactobacillus) and anaerobic (which respire in the absence of oxygen. e.g. Pseudomonas).
12. Reproduction: Bacteria reproduce through asexually by binary fission and endospore
formation and sexually by conjugation. In conjugation, genetic exchange and recombination

occurs through sex pili, but this is a form of horizontal gene transfer and is not a replicative
process, simply involving the transference of DNA between two cells.
 30

Cyanobacteria

The division Cyanophyta includes a large number of Algae which are characterized by a low state
of cell organization

• The cells lack well defined nucleus and characterized by a blue green coloration, the chief
pigments being Chlorophyll a, Carotenes, Xanthophylls, c-Phycocyanin and c- Phycoerythrin

• The product of photosynthesis is glycogen

• These organisms lack flagellated reproductive bodies and sexual reproduction is totally absent

General Characters

The most widely distributed of any group of algae and inhabit marine and freshwater
environments, moist soils and rocks, either as freeliving or as symbiotic organisms Some
planktonic forms can float owing to the presence of gas vacuoles, and most of the filamentous
forms have gliding motility

• Structural range from unicells through branched and unbranched filaments to unspecialized
colonial aggregations which are surrounded by a firm or amorphous mucilage

• Numerically these organisms dominate the ocean ecosystems and there are approximately
1024 cyanobacterial cells in the oceans

• Classified as obligate photoautotrophic organisms

• All blue-green algae are non-motile Gram negative eubacteria They lack a nucleus and
organelles (chloroplast, mitochondria)

• Contain polyhedral bodies (carboxysomes) containing RuBisCo (ribulose bisphospate

carboxylase/oxygenase, the enzyme that converts inorganic carbon to reduced organic carbon
in all oxygen evolving photosynthetic organisms)

• Circular DNA, no chromosomes, no histone protein, 70S ribosomes, smaller than eukaryotic •
Cell walls characterized by a peptidoglycan layer

• Reproduction is strictly asexual, by simple cell division or fragmentation of the colony or

filaments.

Cell organization

• Pigmentation of cyanobacteria includes chlorophyll a, blue and red phycobilins

(phycoerythrin, phycocyanin, allophycocyanin, and phycoerythrocyanin), carotenoids and
xanthophylls
 31

• These accessory pigments lie in the phycobilisomes, located in rows on the outer surface of the
thylakoids. ƒThe traditional name of blue-green algae for the Cyanophyceae is due to the
presence of phycocyanin and phycoerythrin, which usually mask the chlorophyll pigmentation

• Their thylakoids, which lie free in the cytoplasm, are not arranged in stacks, but singled and
equidistant, in contrast to prochlorophytes and most other algae, but similar to Rhodopyta and
Glaucophyta

• The reserve polysaccharide is cyanophycean starch, stored in tiny granules lying between the
thylakoids. In addition, these cells often contain cyanophycin granules, that is, polymer of
arginine and asparagine.

• Some marine species contain gas vesicles used for buoyancy regulation.

• In some filamentous cyanobacteria, heterocysts and akinetes are formed

• Cyanobacteria in extreme habitats show Anoxygenic photosynthesis

• They lack photosystem II perform photosynthesis without O2 production and use hydrogen
sulfide (H2S) as electron donor 2H2S + CO2 → CH2O + 2S + H2O

• Some Thermophilic forms live in extremely high temperature of up to 70°C (Yellowstone Park)

Mycoplasma

Mycoplasmas are typical prokaryotes and are the smallest and simplest self reproducing
microorganisms. They are capable of autonomous growth and reproduction. It is genus of
bacteria lacking a cell wall, hence placed in a separate class Mollicutes. Formerly mycoplasmas
were called as Pleuropneumonia-like organisms (PPLO) (Figure 6). Absence of a cell wall
makes them resistant against many common antibiotics, which target cell wall synthesis. But
Mycoplasma is bounded by a triple-layered unit membrane. Nowak first proposed the term
“Mycoplasma” to replace PPLO.
 32

Mycoplasma are pleomorphic (vary in shape) and mostly non-motile and do not produce spores.
They multiply by binary fission. They can be saprophytic or parasitic and usually facultative
anaerobes. Some species are pathogenic in humans, e.g. M. pneumoniae, causes pneumonia and
other respiratory disorders

In 1960s, some plant diseases were found to be caused by such microorganisms which resemble
mycoplasmas in their morphology and characteristics but differ from Mycoplasmas in not
satisfying “Koch’s postulates”, the pathogenecity test of a pathogen. Due to this, these microbes
have been named “Mycoplasma-like Organism (MLOs).

The smallest viable Mycoplasma cell known is that of Mycoplasma hominis H39 which is about
0.33 micrometer.

Prions

Prions are proteinous infectious particles that lack nucleic acid. Thus, it is entirely different entity
from bacteria, fungus or virus. Stanley B. Prusiner, coined the term “prion” In 1982.

As Prions are misfolded protein molecules, hence not considered as living organisms, which may
propagate by transferring a misfolded protein state. These are extremely stable and gather in
infected tissue, causing tissue damage and cell death.

Prions are composed of an uncharacteristic pathogenic isoform of the prion protein (PrP) in
mammals. PrP found in infectious material has a various structure. The normal form of the
protein is called PrPC, whereas PrPSc is the infectious (the C is for 'cellular' PrP, while the Sc for
'scrapie'). The mammalian prions cause Scrapie and other neurogenerative diseases, e.g. mad cow
disease, Creutzfeldt-Jakob disease, kuru, fatal familial insomnia, and an anomalous form of
genetic dementia. All known prion diseases, collectively called Transmissible Spongiform
Encephalopathies (TSEs), are untreatable and deadly. Bovine Spongiform Encephalopathies
also called as Mad cow disease is a famous example of prions disease. No plant disease caused
by prions is known.

The normal form, PrPC, is converted into PrPsC through a process whereby aportion of its α-
helical and coil-structure is refolded into a β-sheet. This structural transistion is accompained by
profound changes in the physiochemical properties of the PrP. PrPC is sensitive to proteases
whereas PrPsC is proteases resistant. High content of β-sheets in PrPsC results in theformation
of amyloid structure that is absent from the PrPC form
 33

Unit II

CELL ORGANELLES

Introduction:-

Mitochondria (Gr., mito, thread; chondrion, granule) are thread like or granular structures of
eukaryotic cells. These may assume rod-like shape called chondriosomes which may enlarge or
aggregate to form massive spheroidal bodies called chondriospheres. These are not present in
bacterial cells. Mitochondria are the 'power plants' which by oxidation release the energy
contained in the fuel molecules or nutrients and make other forms of chemical energy. The main
function of mitochondria is oxidative phosphorylation, which is an exergonic reaction, meaning
that it releases energy. In prokaryotes, oxidation of organic material is carried out by plasma
membrane enzymes.

Mitochondria - Structure of Mitochondria (A) and Enlarged View ofATP Synthase (B)

Morphology of Mitochondria

Morphologically mitochondria may be in the form of filaments or small granules. These may
assume rod-like shape called chondriosomes which may enlarge or aggregate to form massive
spheroid bodies called chondriospheres
 34

1. Position- Mitochondria lie freely in cytoplasm, possessing power of independent movement

and may take the form of filaments. In some cells they can move freely, carrying ATP where
needed, but in others they are located permanently near the region of the cell where more
energy is needed. E.g., in the rod and cone cells of retina mitochondria are located in the inner
segment, in cells of kidney tubules they occur in the folds of basal regions near plasma
membrane, in neurons they are located in the transmitting region of impulse, in certain muscle
cells (e.g. diaphragm), mitochondria are grouped like rings or bracers around the I-band of
myofibril. During cell division they get concentrated around the spindle.

2. Number- The number of mitochondria varies a good deal from cell to cell and from species to
species. A few algae and some protozoan have only single mitochondria. Their number is related
to the activity, age and type of the cell. Growing, dividing and actively synthesizing cells contain
more mitochondria than the other cells. In Amoeba (Chaos chaos), there may be as many as
50,000 mitochondria. In rat liver cells, these are few in number, about 1000 to 1600. Some
Oocytes contain as many as 3, 00,000 mitochondria.

3. Size- The average size of mitochondria is 0.5-1.0µ in diameter and about 2-8 µ in length. In
exocrine cells of mammalian pancreas they are about 10 µ long and in oocytes of amphibian Rana
pipiens are 20-40µ long. Yeast cells have the smallest mitochondria.

Ultra structure of Mitochondria

1. Membranes- Both the inner and the outer mitochondrial membranes resemble the
plasma membrane in molecular structure. Each of them is 60-70Å, trilamellar and
composed of two layers of phospholipid molecules sandwiched between two layers of
protein molecules. However, the two membranes differ in the kinds of protein and lipids
they have and also in their properties. Both the outer and the inner membranes contain
specific pumps or channels, for the transport of molecules through them. The
membranes may be connected at adhesion sites through which proteins are transferred
from the outer to the inner membrane. The outer and the inner membrane are separated
from each other by a narrow space called the inter-membrane space or outer chamber
or peri-mitochondrial space. It is about 80Å wide. It contains a clear homogeneous fluid.
(i) Outer Membrane- The outer membrane is smooth permeable to most small
molecules, having trans-membrane channels formed by the protein 'porin'. It
consists of about 50% lipid, including a large amount of cholesterol. It contains
some enzymes but is poor in protein.
(ii) Inner Membrane- The inner membrane is selectively permeable and regulates
the movement of materials into and out of the mitochondrion. It is rich in
enzymes and carrier proteins permease. It has a very high protein/lipid ratio
 35

(about 4:1 by weight). It lacks cholesterol. Cardiolipin is closely associated with

certain integral proteins and is apparently required for their activity

2. Matrix- The space between the cristae called the inner chamber is filled with a gel like material
termed the mitochondrial matrix. It contains proteins, lipids, some ribosomes, RNA, one or two
DNA molecules and certain fibrils, crystals and dense granules.

3. Cristae- The inner mitochondrial membrane bears plate like infoldings called the cristae. They
extend inwards to varying degrees, and may fuse with those from the opposite side, dividing the
mitochondrion into compartments. They are arranged in a characteristic manner in different
cells. Normally they run at right angles to the long axis of the rod shaped mitochondria. In cells
of the proximal parts of the kidney tubules, the cristae are longitudinal folds parallel to the long
axis of mitochondrion. In many protozoans, in insect flight muscles cells and in adrenal
endocrine cells the cristae are tubular. Cristae are lamellar in hepatocytes. In heart muscle cells
cristae are zig-zag.

4. Oxysomes- The inner mitochondrial membrane bears minute regularly spaced particles
known as the inner membrane subunits or elementary particles (EP) or oxysomes. An oxysome
consists of three parts- a rounded head piece or F1 subunit joined by a short stalk to a base piece
or F0 subunit located in the inner membrane. There may be 100,000 to 1000,000 oxysomes in a
single mitochondrion.

Biogenesis of Mitochondria:-

 The formation of new mitochondria has been explained with the following hypothesis.
 36

 De Novo Synthesis- According to this hypothesis mitochondria arises de novo from

precursors in the cytoplasm.
 Origin from membrane- This hypothesis proposes that the mitochondria arises from the
invaginations of plasma membrane, endoplasmic reticulum, Golgi apparatus or nuclear
envelop. The membrane invaginates and extends into the cytoplasm as a tubular structure.
It gradually becomes curved and folded and forms a double walled structure, the
mitochondrion.
 Develop from Micro bodies- It is held that they mitochondria are developed by the
accumulation of micro bodies in the cytoplasm. A micro body consists of a single outer
membrane and a dense matrix with a few cristae which eventually develops into fully
formed mitochondria.
 Prokaryotic Origin- It is believed that mitochondria are originated from bacteria. It is
supported by many evidences.
 First is the localization of enzymes of respiratory chain, which in case of bacteria, are
localized in plasma membrane which can be compared with the inner membrane of the
mitochondrion.
 In some bacteria, plasma membrane forms membranous projections (called mesosomes)
like cristae of mitochondria. These mesosomes possess respiratory chain enzymes.
 The mitochondrial DNA is circular as it is in bacteria. Replication process of mitochondria
is similar to bacteria.
 Ribosomes in mitochondria are smaller and similar in size to that of bacterial ribosomes.
 Chloramphenicol inhibits the synthesis of protein in mitochondria as well as in bacteria.
Furthermore, in the process of protein synthesis, mitochondria depend partially on
mitochondrial matrix and DNA and partially on nucleus and cytoplasm of the eukaryotic
cells. It exhibits the symbiotic nature of mitochondria. These evidences support the
prokaryotic origin of mitochondria.
 Replication- It is held that mitochondria are self-replicating organelles. New mitochondria
arise by some type of splitting process from pre-existing mitochondria.
 The last hypothesis seems probable. Since the mitochondria have their own DNA and
ribosomes, they can replicate new mitochondria. However, there is a nuclear control over
the process as the mitochondria synthesize some of their proteins themselves and get
others from the cytoplasm of the cell formed under the direction of the nuclear DNA.

Functions of Mitochondria:-

Cell respiration takes place in mitochondria and so they are known as the 'power house' of the
cell. They bring about stepwise oxidation of food stuffs or "low-grade" fuel of the cell and transfer
 37

the energy so released to the energy carrier ATP, the "high-grade" fuel of the cell. ATP is used to
bring about the energy-requiring activities in the cells, namely, biosynthesis, active transport,
transmission of nerve impulse, muscle contraction, cell growth and division and
bioluminescence.

 Mitochondria provide intermediates for the synthesis of important biomolecules

 such as chlorophyll, cytochromes, steroids etc.
 Some amino acids are also formed in the mitochondria.
 Mitochondria actively accumulate calcium ions as calcium phosphate precipitate. They
regulate the calcium ions concentration in the cytoplasm by storing and releasing Ca+. The
calcium ions regulate numerous biochemical activities in the cell

Endoplasmic Reticulum (ER)

The matrix of cell contains various particles of different sizes called cytoplasmic constituents or
organelles. They include rounded, globular, filamentous or granular mitochondria, network of
endoplasmic reticulum, elongated secretary particles of Golgi apparatus, ribosomes, plastids,
centrosomes and lysosomes. Endoplasmic reticulum is a complex, finely divided vacuolar or tubular
system, extending from nucleus through cytoplasm to the margins of the cells. This system is
enclosed by double membrane. Ribosomes are small dense and granular ribonucleoprotein (i.e. RNA
and proteins) particles found attached to outer surface of endoplasmic reticulum and nucleus as well
as freely scattered in cytoplasm, mitochondrial matrix and chloroplast. Golgi bodies may consist of
many flattened sacs. In plant cells they are collectively called as ‘dictyosome’. They are found
scattered throughout the cytoplasm. Golgi complex occupies different positions in different kinds of
cells. In secretary and absorptive cells, it usually lies between the nucleus and the cell surface where
secretion and absorption occurs. In nerve cells it surrounds the nucleus, and lies elsewhere in other
cells.

In eukaryotic cells endoplasmic reticulum is generally the largest membrane which forms extensive
system of intercommunicating membranous sacs or channels. It represents 30 to 60% of total
membrane in a cell. The membrane of endoplasmic reticulum may or may not have ribosomes
attached to their outer membrane. Accordingly these are classified as rough (RER) or smooth
endoplasmic reticulum (SER). Rough endoplasmic reticulum is characterized by the presence of
ribosomes of about 150Å in diameter and rich in protein and RNA. Smooth endoplasmic reticulum
lacks ribosomes. It comprises three types of elements: cisternae, tubules and vesicles.

Cisternae- These are flattened, unbranched, sac like elements with about 40-50µm in diameter. They
lie in stacks (piles) parallel to but interconnected with one another. They are separated from one
 38

another by cytosolic spaces. The small granular structures called the ribosomes may or may not be
present on the surface of cisternae.

Tubules- These are irregular, branching elements, which form a network along with other elements.
They are about 50-100µm in diameter, and are often free of ribosomes.

Vesicles- These are oval, vacuole like elements, about 25-500µm in diameter. They often occur
isolated in the cytoplasmic matrix. They are also free of ribosomes. A fluid called the endoplasmic
matrix is present in the lumen of ER. All the elements of ER freely communicate with one another

Ultrastructure of Endoplasmic reticulum

The membrane bounding the cisternae, tubules and vacuoles of the ER is similar to the cell
membrane. It is 50-60Å thick. The membranes of endoplasmic reticulum are composed of two layers
of phospholipids molecules sandwiched by two layers of protein molecules like other membranes in
the cell (Robertson, 1959). The ER membrane has a relatively high protein/lipid ratio. It is
continuous with the cell membrane, Golgi membranes and outer membrane of the nuclear envelope.
Certain cisternae open out by pores in the cell membrane. In the lumen of endoplasmic reticulum,
secretary granules were observed by Palade (1956). The lumen acts as a passage for the secretary
products. About 30-40 different enzymes are associated with the ER for the various synthetic
activities. These may be located on the cytoplasmic surface or luminal surface or both. Membrane
bound endoplasmic reticulum spaces varies in shape and sizes in different cell types

On the basis of absence or presence of ribosomes, two kinds of ER are found in cells.

1. Smooth Endoplasmic Reticulum: Ribosomes are absent on the walls of ER and so it appears
smooth and hence called smooth or agranular ER. It mainly occurs as tubular forms. The tubules
forms irregular lattices and measures about 500-1000Å in diameter. Smooth ER is commonly found
in the cells involved in the synthesis of steroids or lipids i.e. non protein type of synthesis
(Christensen and Fawcett, 1961) such as adrenal or sebaceous glands, gonadial interstitial cells.
Certain cells with carbohydrate metabolism (e.g. liver cells), impulse conduction (e.g. muscle cells),
with pigment production (e.g., retinal pigment cell) and electrolyte excretion (e.g., chloride cells of
fish gills) are also have more of SER in them.

2. Rough Endoplasmic Reticulum (RER): It is characterized by the presence of ribosomes on the

surface of reticulum and so it is also known as granular ER. It is in the form of flattened cisternae
with the width of 400-500Å. RER occurs largely in the cells that are actively involved in the synthesis
of proteins such as enzymes (e.g. pancreatic cells, plasma cells and liver cells) or mucus (goblet cells).
In exocrine cells of pancreas, RER consists of reticular sheets and fenestrated cisternae in the basal
region of the cell. These cisternae measures about 5-10 micron in length and their groups are 400-
 39

1000Å in diameter. In apical region of the cells, granular reticulum occurs in the form of vesicles.
Granular and agranular ER are in continuity of their membranes in the regions of contact.

The vesicular network starts from nuclear membrane and spread throughout thecytosol constitutes
endoplasmic reticulum. There are twodifferent types of Endoplasmic Reticulum (ER) present in
the cell, Rough Endoplasmic Reticulum (RER), and Smooth Endoplasmic Reticulum (SER). RER has
ribosome attached to it to give a rough appearance whereas smooth endoplasmic reticulum is
devoid of ribosomes. Protein synthesis on ribosomeattached to RER are sorted into three different
categories, such as integral membrane proteins, proteins for secretion and protein destined for
different organelles. Proteins are synthesized with n-signal peptide and these signal peptides are
recognized by signal recognition particle on theirtarget organelles.For example, if a protein is synthesized
with a signal peptide for mitochondria, it will attach tosignal recognition particle and receptor onto the
outer mitochondrial membrane todeliver the protein. The proteins without any signal peptide tags are
supposed toremain in the cytosol.

Functions of Smooth Endoplasmic Reticulum

1. Surface for Synthesis- The SER provides surface for the synthesis of fatty acids, phospholipids,
glycolipids, steroids and visual pigments.
2. Glycogen Metabolism- The SER carries enzymes for glycogen metabolism in liver cells.
Glycogen granules are attached in larger numbers to the outside of the SER’s membranes in liver
cells.
3. Detoxification- The SER has enzymes that are involved in the detoxification in the liver, i.e.,
converts harmful materials such as carcinogens and pesticides, into harmless ones for excretion
by the cell.
4. Formation of organelles- The SER produces Golgi apparatus, lysosomes, micro bodies and
vacuoles.
5. Transport route- The proteins shift from RER through SER to Golgi apparatus for further
processing.
 40

6. Skeletal Muscle Contraction- The sarcoplasmic reticulum in skeletal muscle cells release Ca2+
ions to cause contraction and absorbs Ca2+ ions to bring about relaxation.
7. Fat Oxidation- The SER membranes carry out the initial reactions in the oxidation of fats.
Functions of Rough Endoplasmic Reticulum
1. Surface for Ribosomes- The RER provides a large surface for the attachment of ribosomes.
2. Surface for synthesis- The RER offers extensive surface on which protein synthesis can be
conveniently carried on by ribosomes. The newly formed proteins may enter the ER membranes,
becoming a part of the membrane structure or pass into the ER lumen. The proteins becoming a
part of ER membrane eventually move from the ER via membranes of other cell organelles,
namely Golgi apparatus, secretary vesicles to become permanent plasma membrane proteins.
The proteins entering ER lumen are packed for export.
3. Packaging- The proteins in ER lumen are processed and get enclosed in spherical membrane
bound vesicles which get pinch off from the ER. These vesicles have various fates. Some remain
in the cytoplasm as storage vesicles while others migrate to the plasma membrane and expel
their contents by exocytosis. Some fuse with Golgi apparatus for further processing of their
proteins for storage or release from the cell.
4. Smooth ER Formation- The RER gives rise to the smooth ER by loss of ribosomes.
5. Formation of Nuclear Envelope- The RER forms nuclear envelope around daughter cells in cell
division.
6. Formation of Glycoproteins- The process of linking sugars to proteins to form glycoproteins
starts in the RER and is completed in Golgi apparatus.

Importance of Endoplasmic Reticulum

1. Transport of Materials- The ER facilitates transport of materials from one part of the cell to
another thus forming the cell's circulatory system.

2. Formation of Desmotubule- Tubular extension, called desmotubule, extends through

plasmodesmata to make ER continuous in the two adjacent plant cells.
 41

3. Support- The ER acts as an intracellular supporting framework, the cytoskeleton that also
maintains the form of the cell.

4. Localization of Organelles- It keeps the cell organelles properly stationed and distributed in
relation to one another.

5. Surface for Synthesis- The ER offers extensive surface for the synthesis of a variety of
materials.

6. Storage of Materials- The ER provides space for temporary storage of synthetic products such
as proteins and glycogen.

7. Exchange of materials- The ER helps in the exchange of materials between the cytoplasm and
the nucleus.

8. Location of Enzymes- A variety of enzymes is located in the ER membranes to catalyze the

biochemical reactions .

Ribosomes

Ribosomes are of two types 70S and 80S. ‘S’ is Svedberg unit, a measure of particle size
dependent on the speed with which the particles sediment in the ultracentrifuge. The 70S
ribosomes are found in the prokaryotic cells and in the mitochondria and plastids of eukaryotic
cells. The 80S ribosomes occur in the cytoplasm of the eukaryotic cells. Both the 70S and 80S
ribosomes are similar in structure. They are small, spherical structures of which 70S ribosomes
are around 200Å in diameter, while 80S are 250 to 300Å in diameter. They are porous and
hydrated having two subunits, one is larger (140-160Å in diameter) having dome shaped
structure and the other is smaller in size, found over the larger subunit, forming a cap like
structure. The two subunits are separated by clefts (Palade and Kuff, 1966). Membrane is absent
around them. The subunits occur separately in the cytoplasm, and join to form ribosomes only
at the time of protein synthesis. Many ribosomes line up and join the mRNA chain. After the
synthesis of protein, the ribosomes leave the mRNA chain and dissociate into subunits.

1. 70S Ribosome: These are found in bacterial cells and have the molecular wt. 2.7 × 10–6 daltons
and sedimentation coefficient 70S. 70S ribosome consists of a large 50S subunit and a small 30S
subunit. Each subunit is composed of rRNA and several basic proteins. The 50S subunit has two
species of RNA: 23S and 5S and about 34 different ribosomal proteins. The 30S subunit has only
one species of rRNA, i.e., 16S and about 21 different ribosomal proteins. They also occur in
mitochondria and chloroplasts of eukaryotic cells.
 42

2. 80S Ribosome: Having the sedimentation coefficient 80S, these are somewhat larger and
contain more RNA and proteins than 70S ribosomes. An 80S ribosome is over 250 to 300Å in
diameter. Their mol. wt. is 4 × 10–6 daltons. It consists of a large 60S subunit and a small 40S
subunit. Each subunit is composed of rRNA and several specific basic proteins. The 60S subunit
has three species of rRNA: 28S, 5.8S and 5S and over 45 different ribosomal proteins. The 40S
subunit has only one species of rRNA, i.e., 18S and over 33 different ribosomal proteins. They
are found in eukaryotic cells.

Ultrastructure of ribosomes

The ribosomes are composed of two subunits (one subunit is almost twice in size than the other)
fitted together to form a complete unit of about 300Å in diameter. In 70S ribosome the 50S
subunit is pentagonal compact particle of 160 to 180Å bearing a round concave area in its center
of about 40 to 60Å that accommodates the small subunit. A small pore like transparent area is
also present that inhibits the entrance of enzyme ribonuclease. Similar pores are present in 60S
subunit of 80S ribosomes. The smaller subunits 30S of 70S and 40S of 80S ribosomes have
irregular forms and are often divided into two portions which are interconnected by a strand of
 43

30 to 60 Å thicknesses. Ribosomes have a groove at the junction of large and small subunits. The
mRNA is seated in the gap between both ribosomal subunits, where the ribosome protects a
stretch of some 25 nucleotides of mRNA from degradation by ribonuclease. From this groove, a
canal or tunnel extends through the large subunit and opens into the lumen of the endoplasmic
reticulum. Polypeptides are synthesized in the groove between the two ribosomal subunits and
pass through the tunnel of the large subunit into the endoplasmic reticulum

Functions of Ribosome

1. Attached Ribosomes- The ribosomes provide space and enzymes for the synthesis of proteins
in the cell. The ribosomes bound to the ER membranes synthesize:

(i) integral proteins for cellular membranes,

(ii) lysosomal proteins and

(iii) secretary proteins for export as secretions.

2. Free Ribosomes- The free ribosomes produce structural and enzymatic proteins for use in the
cell itself. These proteins include glycolytic enzymes and most extrinsic membrane proteins,
such as spectrin.

Importance of Ribosome

Ribosomes are known as protein factories. Ribosomal RNA molecules possibly serve as a skeletal
framework in the ribosomes.

Smaller ribosomal subunit is required for the formation of initiation complex at the start of the
protein synthesis. Whereas larger ribosomal subunit is necessary for peptide bond formation
and the elongation for the polypeptide.

The ribosome function as a template in order to bring together various components involved in
the synthesis of proteins. Ribosomes co-ordinate the interaction of t-RNA- amino acid complex
 44

with m-RNA. This co-ordination results in the translation of genetic code forming specific
proteins.

Since free ribosomes are not involved in protein synthesis, they are transported through
endoplasmic reticulum membranes and assembled into globules within the cisternae and canals
in the cells that produce 'proteins for transport'. Proteins later appear in the form of granules
outside the Golgi complex.

GOLGI APPARATUS

The Golgi apparatus, like the endoplasmic reticulum, is a canalicular system withsacs that performssome
important cellular functionslikebiosynthesis of polysaccha rides and packaging of cellular products.

In 1898, an Italian biologist, Camillo Golgi discovered a darkyellow networklocated near the nucleus
of nerve cells. This network, which was later identified in other cell types, was named the Golgi complex
or Golgi body. Since originally these were known to be networks, they were also called
‘dictyosomes’(Gr., dictyes = net).

The Golgi complex occur in all cells except the prokaryotic cells and someeukaryotic cells like
mature sieve tubes of plants, mature sperm and red blood cells of animals. In animal cells, there
usually occurs a single Golgi apparatushowever, its number may vary.

Thus, Paramoeba species has two Golgi apparatuses, and nerve cells, liver cells and chordate
oocytes have multiple Golgi apparatuses. In animal cells, theGolgi apparatus is a localized organelle.
Usually it remains polar and occurs in-between the nucleus and the periphery (for example, thyroid
cells, goblet cells).

In nerve cells it occupies a circum-nuclear position. However, in the cells of higher plants, the Golgi
bodies or dictyosomes are usually found scattered through-out the cytoplasm.
 45

Functions of Golgi Complex

Golgi apparatus is metabolically very active. Many functions have been assigned to it:

1. Formation of secretary vesicles- The Golgi complex processes and packages proteins and
lipids coming from the ER for transport to other parts of the cell or out of the cell. Packaging
involves wrapping the materials by a membrane, forming secretary vesicles. The materials so
packed includes zymogen in pancreatic cells, mucus in goblet cells, lactoprotein in mammary
gland cells, pigment granules in pigment cells, collagen in connective tissue cells, hormones in
endocrine cells, etc.

2. Synthesis of carbohydrates- The Golgi apparatus synthesizes certain mucopolysaccharides

from simple sugars.

3. Formation of Glycoproteins- The Golgi apparatus links the sugars with proteins coming from
rough ER to form glycoproteins.

4. Formation of Lipoproteins- Lipids and proteins coming from the ER are complexed into
lipoproteins in the Golgi apparatus.

5. Addition to Cell Membrane- The Golgi apparatus provides membrane material for the plasma
membrane when the later must enlarge for the formation of pinocytotic and phagocytotic
vesicles and for the formation of cleavage furrow during the division of animal cells. As the
secretary vesicles discharge their contents by exocytosis, their membranes are incorporated
into the cell membrane. This enlarges the cell membrane. Since, endocytosis removes segments
of the cell membrane, the latter's enlargement by exocytosis is temporary, rather compensatory.
The transfer of membrane from the ER via transition vesicles, Golgi complex and secretary
vesicles to the plasma membrane is called membrane flow.

6. Membrane Transformation- The Golgi apparatus changes one type of membrane into another
type. Membranes are gradually modified from the ER type to one with characteristics of the
plasma membrane as they shift through the Golgi complex.

7. Formation of cell wall- In some algae, cellulose plates for cell wall is synthesized in Golgi
complex. In higher plants the Golgi complex (a) synthesizes pectin and some carbohydrates
necessary for the formation of cell wall and (b) produces some secretions such as mucilage,
gums, etc.

8. Formation of lysosomes- The Golgi complex gives rise to primary lysosomes by budding. The
lysosomes may also arise from ER.

9. Acrosome Formation- The Golgi complex gives rise to the acrosome in a sperm.
 46

10. Formation of Yolk and Cortical Granules- The Golgi complex produces yolk and cortical
granules in the eggs. Formation of yolk is called vitellogenesis.

11. Formation of Nematocysts and Trichocysts- The Golgi apparatus gives rise to the
nematocysts in Hydra and perhaps also in other coelenterates, and trichocysts in ciliates such as
Paramecium.

12. Storage of Secretions- The Golgi complex stores cell secretions such as proteins and lipids.

13. Absorption of Materials- Golgi apparatus absorbs materials from the environment. For
example, cells of the intestinal lining use Golgi apparatus to absorb lipids from the intestine.

14. Location of Enzymes- A variety of enzymes is localized in the Golgi complex to help in the
cell's biochemical reactions.

The Golgi apparatus is often referred to as the "traffic police" of the cell because its enzymes sort
out and modify cell's secretary proteins passing through its lumen and membrane proteins in its
membranes and directs them to their proper destination.

Plastids:

 Plastid is a double membrane-bound organelle involved in the synthesis and storage of

food, commonly found within the cells of photosynthetic plants.

 Plastids were discovered and named by Ernst Haeckel, but A. F. W. Schimper was the first
to provide a clear definition.
 They are necessary for essential life processes, like photosynthesis and food storage.
 A plastid containing green pigment (chlorophyll) is called chloroplast whereas a plastid
containing pigments apart from green is called a chromoplast. A plastid that lacks
pigments is called a leucoplast and is involved mainly in food storage.

Types of Plastids
 47

An undifferentiated plastid is called a proplastid. It may develop later into any of the other
plastids.
A. Chloroplasts
 The chloroplasts are probably the most-known of the plastids.
 These are responsible for photosynthesis.
 The chloroplast is filled with thylakoids, which is where photosynthesis occurs, and
chlorophyll remains.
B. Chromoplasts
 Chromoplasts are units where pigments are stored and synthesized in the plant.
 These are found in flowering plants, fruits, and aging leaves.
 The chloroplasts actually convert over to chromoplasts.
 The carotenoid pigments allow for the different colors seen in fruits and the fall leaves.
One of the main reasons for these structures and the colors is to attract pollinators.
C. Leucoplasts
 Leucoplasts are the non-pigmented organelles.

 They are found in the non-photosynthetic parts of the plant, such as the roots.
 Depending on what the plant needs, they may become essentially just storage sheds for
starches, lipids, and proteins.
 They are more readily used for synthesizing amino acids and fatty acids.
 A leucoplast may be an amyloplast that stores starch, an elaioplast that stores fat, or a
proteinoplast that stores proteins.
 48

D. Gerontoplasts
 Gerontoplasts are basically chloroplasts that are going through the aging process.

 These are chloroplasts of the leaves that are beginning to convert into different
organelles or are being re-purposed since the leaf is no longer utilizing photosynthesis
(such as in the fall months).
Structure of Plastids
 Chloroplasts may be spherical, ovoid, or discoid in higher plants and stellate, cup-shaped,
or spiral as in some algae.

 They are usually 4-6 µm in diameter and 20 to 40 in number in each cell of higher plants,
evenly distributed throughout the cytoplasm.
 The chloroplast is bounded by two lipoprotein membranes, an outer and an inner
membrane, with an intermembrane space between them.
 The inner membrane encloses a matrix, the stroma which contains small cylindrical
structures called grana. Most chloroplasts contain 10-100 grana.

 Each granum has a number of disc-shaped membranous sacs called grana lamellae or
thylakoids (80-120Å across) piled one over the other.
 The grana are interconnected by a network of anastomosing tubules called inter-grana or
stroma lamellae.
 Single thylakoids, called stroma thylakoids, are also found in chloroplasts.
 Electron dense bodies, osmophilic granules along with ribosomes (70S), circular DNA,
RNA and soluble enzymes of Calvin cycles are also present in the matrix of the stroma.
 Chloroplasts thus have three different membranes, the outer, the inner and the thylakoid
membrane.

 The thylakoid membrane consists of lipoprotein with a greater amount of lipids which are
galactolipids, sulpholipids, phospholipids.
 The inner surface of the thylakoid membrane is granular in the organization due to small
spheroidal quantosomes.
 49

 The quantosomes are the photosynthetic units, and consist of two structurally distinct
photosystems, PS I and PS II, containing about 250 chlorophyll molecules. Each
photosystem has antenna chlorophyll complexes and one reaction center in which energy
conversion takes place. In higher plants, the pigments present are chlorophyll-a,
chlorophyll-b, carotene, and xanthophyll.
 The two photosystems and the components of the electron transport chain are
asymmetrically distributed across the thylakoid membrane. Electron acceptors of both PS
I and PS II are on the outer (stroma) surface of the thylakoid membrane. Electron donors
of PS I are on the inner (thylakoid space) surface.

Funcitons
All plant cells contain plastids in some shape or form. This roll-call indicates their functional
diversity and demonstrates that plastids lie at the very core of plant cellular function.
 Plastids are the site of manufacture and storage of important chemical compounds used
by the cells of autotrophic eukaryotes.
 The thylakoid membrane contains all the enzymatic components required for photosyn-
thesis. Interaction between chlorophyll, electron carriers, coupling factors, and other
components takes place within the thylakoid membrane. Thus the thylakoid membrane
is a specialized structure that plays a key role in the capture of light and electron
transport.

 Thus, chloroplasts are the centers of synthesis and metabolism of carbohydrates.

 They are not only of crucial importance in photosynthesis but also in the storage of
primary foodstuffs, particularly starch.
 Its function largely depends on the presence of pigments. A plastid involved in
food synthesis typically contains pigments, which are also the ones responsible for the
color of a plant structure (e.g. green leaf, red flower, yellow fruit, etc.).

 Like mitochondria, plastids have their own DNA and ribosomes. Hence, they may be
used in phylogenetic studies.
Centrosomes
Centrosomes are cellular components that appear like a disc in shape and are internally
built by microtubule components and play important role in cell division.
 Centrosomes are double or a pair of centrioles along with microtubule-organizing center
(MTOC) with the pericentriolar matrix.
 The centrioles in the centrosome are arranged in pairs and perpendicularly i.e. forming
the right angle between them.
 Centrioles are present at the base of cilia and flagella so they are also called basal bodies.
 The centrosome is present in an animal cell, not the plant cell or fungi. The plant cell
contains a microtubule-organizing center.
 But they are also present in male gametes of bryophytes, seedless vascular plants,
charophytes, ginkgo, and cycads.
 50

 Development of cancer can occur in the case of non-functional centrosomes in animals.

 It is present in the cytoplasm at the central region i.e. near of nucleus hence called
centrosome or centrioles.

Centrosome structure
 Centrosome is composed of microtubules that are short i.e. length of about 500 nm and
their width or diameter of about 200 nm.
 They are arranged in nine sets (in each centriole) where each set comprises three
microtubules or tubulins which is also termed as 9+3 arrangement.
 The two centrioles are oriented in 90 degrees to one another.
 The centrioles are surrounded by the pericentriolar matrix.
 The pericentriolar matrix comprises dense cytosolic structures and proteins such as
gamma-tubulins, pericentrin, ninein, etc.
 The centrioles and pericentriolar matrix as a whole are called the centrosome.
 The arrangement of microtubules in the centrosome is called cart-wheel structure.
 In the cart-wheel model, the nine microtubules(triplets) linked with each other are
arranged in a circle and there is one microtubule(duplet) at the center connected to the
nine microtubules by proteins called spokes and this arrangement appears like a
cartwheel.


Centrosome function
 51

1. They take part in cell division for the formation of spindle fibers necessary for the
movement of chromosomes towards opposite poles during the separation of sister
chromatids.
2. They are also involved in the cellular organization i.e. by the formation or use of
microtubules to prepare cytoskeleton.

3. Its position in the cell determines the position of other organelles and the nucleus as it is
involved in the formation of the cytoskeleton.
Centrosome in cell division
Each cell contains a centrosome near the nucleus. As the separation of sister chromatids requires
two centrosomes arranged at two opposite poles the centrosome goes through a division
process during cell division.

 In the G1 phase of cell division, initiation of duplication begins in which the centrioles
which were arranged in pairs get separated.
 In the S phase, another centriole formation starts from each mother centriole.
 The complete formation of two centrosomes from a single mother centriole gets
completed in the G2 phase of cell division.
 The centrosomes then get separated and move towards opposite poles of the nucleus
during metaphase after the disintegration of the cell membrane.

Lysosome
Lysosomes are membrane-bound, dense granular structures containing
hydrolytic enzymes responsible mainly for intracellular and extracellular digestion.

 The word “lysosome” is made up of two words “lysis” meaning breakdown and “soma”
meaning body.
 52

 It is an important cell organelle responsible for the inter and extracellular breakdown of
substances.

 They are more commonly found in animal cells while only in some lower plant groups (
slime molds and saprophytic fungi).

 Lysosomes occur freely in the cytoplasm. In animals, found in almost all cells except in
the RBCs.

 They are found in most abundant numbers in cells related to enzymatic reactions such
as liver cells, pancreatic cells, kidney cells, spleen cells, leucocytes, macrophages, etc.

Structure of Lysosomes
 Lysosomes are without any characteristic shape or structure i.e. they are pleomorphic
 They are mostly globular or granular in appearance.

 It is 0.2-0.5 μm in size and is surrounded by a single lipoprotein membrane unique in

composition.
 The membrane contains highly glycosylated lysosomal associated membrane
proteins (LAMP) and Lysosomal integral membrane proteins (LIMP).
 LAMPs and LIMPs form a coat on the inner surface of the membrane
 They protect the membrane from attack by the numerous hydrolytic enzymes retained
inside.
 The lysosomal membrane has a hydrogen proton pump which is responsible for
maintaining pH conditions of the enzyme The acidic medium maintained by the proton
pump that pumps H+ inside the lumen, ensures the functionality of the lysosomal
enzymes.
 Inside the membrane, the organelle contains enzymes in the crystalline form.

Lysosomal Enzymes
 53

For degradation of extra and intracellular material, lysosomes filled with enzymes called
hydrolases. It contains about 40 varieties of enzymes which are classified into the following
main types, namely:
 Proteases, which digest proteins

 Lipases, which digests lipids

 Amylase, which digests carbohydrates
 Nucleases, which digest nucleic acids

 Phosphoric acid monoesters

Collectively the group of enzymes is called hydrolases which cause cleavage of substrates by the
addition of water molecules. Most of the lysosomal enzymes function in the acidic medium.
Lysosomal Membrane

 As compared to the mitochondria, it is slightly thicker.

 Sialic acid is present in it.
 Since the lysosomal membrane protein is highly glycosylated, it protects from the action
of the lysosomal proteases.
 The lysosomal membrane can fuse with the other membranes of the cell which is the
unique property.
 When the lysosomal membrane ruptures, lysosomal enzymes are released.
 It can be caused by the destabilizing influence of the surface-active agents and the
steroid sex hormones.
 The lysosomal membrane is stabilized by cortisone and hydrocortisone.
 On the tissue, they possess an anti-inflammatory effect.
 Within the lysosome all the process of digestion takes place.
 For the action of the lysosomal enzyme, the medium should be acidic.

 To maintain the acidic condition inside the organelle, there should be an accumulation
of the H+.
 It is maintained by the proton pump which is ATP-dependent.
 Transport protein is also present in the lysosomal membrane.

 When the macromolecules get digested, the final products can be transported by these
proteins.
 After the transportation, they can be further utilized by the cell or be excreted.
Types of Lysosomes

 Polymorphism can be seen in the morphology of the lysosome.

 There are four types of lysosomes. They are:
 Primary lysosome
 54

 Heterophagosomes
 Autophagososomes

 Residual Bodies
A. Primary Lysosomes
 They are also called:

 Storage granules
 Protolysosomes
 Virgin lysosomes

 The primary lysosome is bounded by a single membrane.

 It has a diameter of 100nm.
 A digestive enzyme is present in it which has not taken part in the digestion.

 One type of enzyme or another is present in it.

 Only in the secondary lysosome, there is the presence of the full complement of acid
hydrolases.
B. Heterophagosomes
 They are also called:

 heterophagic vacuoles
 heterolysosomes
 Phagolysosomes
 When the primary lysosome fuse with the cytoplasmic vacuoles, a heterophagosome is
formed.
 Extracellular substances are present in the cytoplasmic vacuoles.
 Different endocytic processes like pinocytosis, phagocytosis, or receptor-mediated
endocytosis help in bringing such extracellular substances into the cell.
 In these secondary lysosomes, there is the presence of the hydrolytic enzymes which
digest the engulfed substances.
 After digestion, such particles will pass across the membrane of the lysosome.
 Then it will become part of the matrix.

C. Autophagosomes
 They are also called:
 Autophagic vacuole

 cytolysosomes
 Autolysosomes
 55

 Digestion of the different intracellular structures like mitochondria, ribosome,

peroxisome, and glycogen granules can be done by the primary lysosome.
 Autophagy is called autodigestion.

 During cell growth and repair, autophagy is a normal event.

 It is prevalent in the differentiation, the dedifferentiation of the tissues and tissue under
stress.
Autophagy occurs in different forms:

a. By fusion of the lysosome:

 It is enclosed inside the double membrane sac.
 Then there is the breakdown of the inner membrane.

 Penetration of the enzyme can occur to the enclosed organelle.

b. Formation of a vesicle and fusion with primary lysosome:
 Vesicle is formed when the smooth endoplasmic reticulum encases the organelle which
needs to be digested.
Microautophagy also takes place. When the digestion process proceeds then it becomes difficult
to identify the type of secondary lysosome if it is heterophagosome or autophagosome. So, in
this stage, it is said as the digestive vacuole.
D. Residual Bodies
 They are also called:
 Telolysosome

 Dense bodies
 Incomplete digestion results in the formation of the residual bodies.
 When some lysosomal enzymes are absent, incomplete digestion may occur.

 Inside the digestive vacuoles, the undigested food remains as the residue.
 Then they make take different forms.
 Residue body is larger and irregular in shape.

 By the defecation, residual bodies are eliminated in the case of Amoeba and some other
protozoa.
 In some cells, for a longer period, the residual body may stay which may cause aging.
Functions of Lysosomes
Lysosomes serve two major functions:
1. Intracellular Digestion
 To digest food, the lysosome membrane fuses with the membrane of food vacuole and
squirts the enzymes inside.
 56

 The digested food then diffuses through the vacuole membrane and enters the cell to be
used for energy and growth.
2. Autolytic Action

 Cell organelles that need to be ridden are covered by vesicles or vacuoles by the process
of autophagy to form autophagosome.
 The autophagosome is then destroyed by the action of lysosomal enzymes.
Processes in which lysosomes play crucial roles include:

a. Heterophagy
The taking into the cell of exogenous material by phagocytosis or pinocytosis and the digestion
of the ingested material after fusion of the newly formed vacuole with a lysosome.
b. Autophagy

A normal physiological process that deals with the destruction of cells in the body. It is essential
for maintaining homeostasis, for normal functioning by protein degradation, turnover of
destroyed cell organelles for new cell formation
c. Extracellular Digestion
Primary lysosomes secrete hydrolases outside by exocytosis resulting in degradation of
extracellular materials.
Eg. Saprophytic fungi
d. Autolysis
It refers to the killing of an entire set of cells by the breakdown of the lysosomal membrane. It
occurs during amphibian and insect metamorphosis.
e. Fertilization

The acrosome of the sperm head is a giant lysosome that ruptures and releases enzymes on the
surface of the egg. This provides the way for sperm entry into the egg by digesting the egg
membrane.

PEROXISOMES

In addition to lysosomes, a group of smaller particles than mitochondria andlysosomesare found in

liver cells.These particles are rich in the enzymes peroxidase, catalase, D-amino acid oxidase and to a
lesser extent, urate oxidase and arecalled peroxisomes .
 57

Peroxisome

Peroxisomes are found in liver, kidney, Protozoa, yeast and many cell types of higher plants.
Peroxisomes present in plant cells show some morphological similarities tothe peroxisomes in animal
cells. But plant peroxisomes have different enzymes including the enzymes of the glyoxylate cycle.
Hence their name isglyoxysomes.

Structure

Peroxisomes are ovoid granules limited by a single membrane. They contain afine, granularsubstance
which may condense in the centre, forming an opaque and homogeneous core or nucleoid.The average
size of the peroxisomes in rat livercells was shown to be 0.6 to 0.7 µm. The number of peroxisomes per
cell varied between 70 and 100, whereas 15 to 20 lysosomes were found per liver cell. In many
tissues’ peroxisomes show a crystal-like body made of tubular subunits. In contrast to the nucleoid-
containing peroxisomes found in liver and kidney, there are others which are smaller and lack a
nucleoid. These are called micro peroxisomes found in all cells and are related to endoplasmicreticulum.
These may be considered as regions of ER in which catalase and other enzymes arefound.

Origin

Both types of peroxisomes are formed in the endoplasmic reticulum, and theenzymes they contain
are synthesized by ribosomes bound to the granular ER. It isassumed that the peroxisome grows and is
destroyed, probably by autophageafter four or five days.

Peroxisomal enzymes

The enzymes of peroxisomes are synthesized in the ribosomes attached to therough endoplasmic
reticulum. Liver peroxisomes contain four enzymes related tothe metabolism of H2O2. Three of them are
 58

urate oxidase, D-amino oxidase, and oc-hydroxylic acid oxidase, which produce peroxide (H2O2), and
fourth is catalasewhich destroys peroxide.

Catalase is found in the matrix of peroxisomes and represents up to 40% ofthe total protein. H2O2 is
toxic so catalase destroys it and it probably plays aprotective role. The enzyme urate oxidase, D-
amino oxidase and hydroxylic acidoxidase present in amphibian and avian peroxisomes are related to
the catabolismof purines.

Functions of Peroxisomes

Peroxisomes derive their name from their use of molecular oxygen for metabolic processes. These
organelles are largely associated with lipid metabolism and the processing of reactive oxygen species.
Within lipid metabolism, peroxisomes mostly deal with â–oxidation of fatty acids, the mobilization of
lipid stores in seeds,cholesterol biosynthesis and steroid hormone synthesis.

–oxidation

The main reason for the high energydensityof fats is the low proportion of oxygenatomsin everyfattyacid
molecule. Forinstance,palmitic acid,a fattyacid containing16 carbon atoms and having a molecular mass
of over 250 gms/mole, has onlytwo oxygen atoms. While this makes lipids good storage molecules,
they cannotbedirectlyburnet as fuel or quicklycatabolized in the cytoplasm through glycolysis. They need
to be processed before they can be shunted into the mitochondria forcomplete oxidation through the
citric acid cycle and oxidative phosphorylation.

When these molecules need to be oxidized to release ATP, they need to be first broken down into
smaller molecules before they can be processed in themitochondria. Within peroxisomes, long chain
fattyacidsareprogressivelybrokendown togenerate acetyl coenzymeA(acetyl CoA) in a process called –
oxidation.Acetyl CoA then combines with oxaloacetate to form citrate. While most carbohydrates
enter the citric acid cycle as a three-carbon molecule called pyruvatewhich is then decarboxylated to form
acetyl CoA, peroxisomal –oxidation allowsfatty acids to access the citric acid cycle directly.

One of the main by products of –oxidation is hydrogen peroxide whichcan be harmfulfor the cell. This
molecule is also carefullydetoxified by the enzymecatalase within peroxisomes.

Lipid Biosynthesis and Detoxification

In animal cells, peroxisomes are the sites for some amount of lipid biogenesis, especially of special
phospholipids called plasmalogens that form the myelin sheath in nerve fibres. Peroxisomes are also
necessary for the synthesis of bile salts. About 25% of the alcohol we consume is oxidized to
acetaldehyde in theseorganelles. Their role in detoxifying and oxidizing a number of molecules, metabolic
by products and drugs makes them a prominent part of kidney and liver cells.

Disorders Relating to Peroxisome Function

 59

Disorders arising from deficient peroxisome function could arise from defects in peroxisome
biogenesis, mutated peroxisomal enzymes, or non-functional transporters that recognize PTS1
and PTS2 in cytoplasmic proteins. The most severe of these are rare genetic disorders that result in
impaired brain development and neuronal migration, along with myelin deficiency. Other organs affected
includethe skeletal system, liver, kidney, eyes, heart and lungs.

These disorders are usually caused by mutations in PEX genes, which are necessary for organelle
biogenesis – from the formation of the subcellular membrane,to the recognition of cytoplasmic proteins
and their import into the matrix of theorganelle. For instance, PEX16 is involved in the synthesis of
peroxisomalmembranes, while PEX2 forms the translocation channel for the import of matrixproteins.
PEX5, on the other hand is the receptor for recognizing the PTS1 signalsequence.

Defects in these proteins can cause the accumulation of long chain fattyacids in blood plasma or urine
as well as the inappropriatepresence of phospholipids like plasmalogens in red blood cells.

Nucleus: -

Nucleus is usually the most conspicuous organelle of eukaryotic cell. However, well defined
nucleus is absent in prokaryotic cells. Nucleus is the repository of genome and the source of
informational macromolecules that govern the synthetic activities of the cytoplasm. It is
surrounded by a bilaminary nuclear envelop having pore complexes that permit the nuclear-
cytoplasm transport of materials. In the animal cells, it generally lies in the centre, surrounded
on all sides by the cytoplasm. However, in plant cells it is often pushed to oneside of the cell
due to the presence of large central sap vacuole.

The shape of nucleus is variable according to cell type. It is generally spheroid but ellipsoid or
flattened nuclei may also occur in certain cells. In certain WBC (white blood cells) the nucleus is
dumbbell shaped. In human neutrophil it is trilobed.

Most cells contain a single nucleus, known as mono or uninucleate cells. Cells with two nuclei
are known as binucleate cells e.g. Paramecium. Sometimes more than two nuclei are present in a
single cell. Such cells are called polynucleate or multinucleated cells. Such cells in animals are
called syncytial cells (e.g. osteoblast) and such plants are termedcoenocytes (e.g. siphonal algae).
Cells having distinct nucleus are called eukaryotic cells, whereas cells without definite nucleus
are called prokaryotic cells (e.g. bacteria). The latter possess scattered chromatin material
(DNA) in the cytoplasm called nucleoid. The mature mammalian erythrocytes also do not
possess any nucleus.

Size of nucleus is not constant and is generally correlated with DNA content. The nuclear size is
variable depending upon the number of chromosomes (DNA content).
 60

Structure of Nucleus:-

The nucleus consists of various parts. It is bounded by a thin but clearly defined covering, the
nuclear envelop or karyotheca. Within the envelope is a clear fluid substance called nucleoplasm
or nuclear sap or karyolymph is present in which the solutes of the nucleus are dissolved.
Suspended in the nucleoplasm are network of protein-containing fibrilscalled nuclear matrix;
fine intermingled nucleoprotein filaments collectively referred to as thechromatin; and one or
more spherical bodies known as nucleoli (singular, nucleolus). There are no membranes or
microtubules inside the nucleus. Protozoans that form a mitotic spindle within the nuclear
envelop, however, have microtubules in their nuclei.

Structure of nucleus

Chemical Composition:

The nucleus is composed of about 9-12% DNA, 5% RNA, 3% lipids, 15% simple basic proteins
such as histone or protamines, about 65% complex acid or neutral proteins, including enzymes
such as polymerases for the synthesis of DNA and RNA, organic phosphates and inorganic salts
or ions such as Mg++, Ca++ and Fe++.

Functions:

 The nucleus acts as a control center of the cell. It serves the following main functions:
 It maintains the cell by directing the synthesis of structural proteins.
 It regulates cell metabolism by directing the synthesis of enzymatic proteins.
 It contains genetic information for reproduction, development and behavior of the
organism besides for structure and metabolism.
 It brings about cell replication when needed.
 It is the site for the formation of ribosome subunits.
 61

 It brings about cell differentiation by keeping only certain genes operational.

 It develops genetic variations that result in evolution.

Nucleolus (Little Nucleus):-

The nucleolus was discovered in 1781 by F. Fontana in the slime from the eel skin. It is present
in the nucleus of most cells, but is inconspicuous or absent in sperm cells and in muscle cells. It
is usually spherical, but may have other forms. The number of nucleoli in a nucleus varies in
different species. The nucleoli disappear during cell division, and are reformed at specific
sites, the nucleolar organizers or nucleolar organizer regions (NORs), of certain chromosomes,
the nucleolar chromosomes, at the end of cell division before the chromosomes become diffuse.
Position of the nucleolus in the nucleus is often eccentric. However, it occupies a specific position
on its chromosome.

The nucleolus is a dense, somewhat rounded, dark staining organelle. It is without a limiting
membrane. Calcium ions keep it intact. It consists of four regions.

Fibrillar Region or Nucleolonema- It contains indistinct fibrils about 50-100Å in diameter. The
fibrils represent the long rRNA precursor molecules in early stages of processing before the
processing enzymes have cut off segments from them.

Granular Region- It contains spherical, electron dense particles, about 150-200 Å in diameter
and with fizzy outline. The granules are ribosomal subunits (rRNA + ribosomal proteins) that
are nearly ready for transport to the cytoplasm.

Amorphous Region or Pars Amorpha- It is a structure-less proteinaceous matrix in which the

granular and fibrillar regions are suspended.

Nucleolar Chromatin- It consists of 100 Å thick chromatin fibers. The latter are a part of the
nucleolar chromosome which follows a tortuous path through the granular and fibrillar
components of the nucleolus. This part contains many copies of DNA that directs the synthesis
of ribosomal RNA. The rest of the nucleolar chromosomelies in the nucleoplasm.

Functions-

 The nucleolus synthesizes and stores rRNA.

 It also stores ribosomal proteins received from the cytoplasm.
 It forms ribosomal subunits by wrapping the rRNA by ribosomal proteins. The ribosomal
subunits pass out through the nuclear pores into the cytoplasm. Here the subunits join to
form ribosomes when needed. Thus, it is the nucleolus which provides machinery
(ribosomes) for protein synthesis.
 The nucleolus also plays a role in cell division.
 62

Importance of Nucleus:

The nucleus is the control center of a cell. It regulates all metabolic activities of the cell and stores
entire hereditary information. A cell without nucleus cannot survive.

Endomembrane system

The endomembrane system is a complex and dynamic network of membranes within eukaryotic
cells that work together to modify, package, and transport lipids and proteins. It plays a crucial
role in maintaining cellular function and organization. Here's a detailed account of the
endomembrane system, including its components and their functions:

Components of the Endomembrane System

1. Nuclear Envelope

- The nuclear envelope surrounds the nucleus and consists of two lipid bilayer membranes: an
inner and an outer membrane. It is perforated by nuclear pores, which regulate the exchange of
substances between the nucleus and the cytoplasm. The nuclear envelope is continuous with the
endoplasmic reticulum.

2. Endoplasmic Reticulum (ER)

- Rough ER (RER): Studded with ribosomes on its cytoplasmic surface, the RER is involved in
the synthesis of proteins destined for secretion, insertion into membranes, or lysosomal use.
These proteins are translocated into the lumen of the RER, where they undergo folding and post-
translational modifications.

- Smooth ER (SER): Lacks ribosomes and is involved in lipid synthesis, detoxification of drugs
and poisons, and calcium ion storage. The SER also metabolizes carbohydrates.

3. Golgi Apparatus

- The Golgi apparatus consists of flattened membrane-bound sacs called cisternae. It modifies,
sorts, and packages proteins and lipids received from the ER. Proteins and lipids are processed
through the cis (entry) face, medial cisternae, and trans (exit) face, where they undergo further
modifications such as glycosylation and are then sorted and shipped to their final destinations.

4. Lysosomes

- Lysosomes are membrane-bound organelles containing hydrolytic enzymes that digest

macromolecules. They are involved in breaking down cellular waste, pathogens, and cellular
debris through processes like autophagy and phagocytosis. The enzymes within lysosomes are
synthesized in the RER and modified in the Golgi apparatus before being transported to the
lysosome.
 63

5. Vacuoles

- Vacuoles are large vesicles derived from the ER and Golgi apparatus. In plant cells, the central
vacuole plays a key role in maintaining turgor pressure, storing nutrients, and degrading waste
products. In animal cells, vacuoles are smaller and primarily involved in storage and transport.

6. Vesicles

- Vesicles are small, membrane-bound sacs that transport substances between various
components of the endomembrane system and the plasma membrane. They play a crucial role
in endocytosis (uptake of external substances) and exocytosis (release of substances from the
cell).

7. Plasma Membrane

- The plasma membrane is the outermost membrane of the cell, composed of a lipid bilayer
with embedded proteins. It regulates the entry and exit of substances, communicates with the
external environment, and provides structural support. The plasma membrane is also involved
in cell signaling and adhesion.

Functions of the Endomembrane System

1. Protein and Lipid Synthesis

- The ER (both rough and smooth) is the site of synthesis for proteins and lipids. Proteins
synthesized on the RER are often destined for secretion or incorporation into cellular
membranes, while lipids synthesized in the SER are essential for membrane structure and
function.

2. Protein Processing and Modification

- Proteins synthesized in the RER are folded and undergo post-translational modifications,
such as glycosylation and disulfide bond formation. The Golgi apparatus further modifies these
proteins, adding complex carbohydrates and targeting them for specific destinations.

3. Transport and Sorting

- The endomembrane system is responsible for transporting proteins and lipids to their correct
locations within the cell. Vesicles shuttle these molecules between the ER, Golgi apparatus,
lysosomes, vacuoles, and the plasma membrane, ensuring they reach their intended
destinations.

4. Degradation and Recycling

 64

- Lysosomes break down macromolecules, damaged organelles, and foreign particles through
enzymatic digestion. The resulting molecules can be recycled and used for new cellular
components, maintaining cellular homeostasis.

5. Detoxification

- The SER plays a crucial role in detoxifying harmful substances, such as drugs and metabolic
byproducts. This is particularly important in liver cells, where the SER is abundant.

6. Storage

- Vacuoles in plant cells store nutrients, waste products, and other substances. They also
maintain osmotic balance and provide structural support through turgor pressure.

Concept of compartmentalisation

Cell compartmentalization is a fundamental organizational principle in eukaryotic cells, allowing

them to maintain distinct microenvironments and efficiently carry out complex biochemical
processes. This concept can be explained in detail by examining the various compartments
within a cell, their functions, and the mechanisms by which compartmentalization is achieved.

1. Definition and Importance

Cell compartmentalization refers to the segregation of different cellular processes into distinct
membrane-bound organelles. This separation allows for specific biochemical environments to
be maintained within the cell, which is crucial for the efficiency and regulation of cellular
functions. Compartmentalization helps in:

- Isolation of incompatible processes: By keeping certain biochemical reactions in separate

compartments, the cell can prevent interference between incompatible processes.

- Increased efficiency: Enzymatic reactions and other cellular processes are optimized within
specialized environments.

- Regulation and control: Cellular processes can be better regulated when confined to specific
compartments.

2. Major Compartments and Their Functions

Nucleus

- Structure: Surrounded by the nuclear envelope, a double membrane with nuclear pores.

- Function: Houses the cell’s genetic material (DNA) and is the site of transcription (DNA to RNA).

Endoplasmic Reticulum (ER)

 65

- Rough ER: Studded with ribosomes; involved in protein synthesis and modification.

- Smooth ER: Lacks ribosomes; involved in lipid synthesis, detoxification, and calcium storage.

Golgi Apparatus

- Structure: Stacks of flattened, membrane-bound cisternae.

- Function: Modifies, sorts, and packages proteins and lipids for secretion or delivery to other
organelles.

Mitochondria

- Structure: Double-membraned organelles with an inner membrane folded into cristae.

- Function: Powerhouse of the cell; site of ATP production through cellular respiration.

Lysosomes

- Structure: Membrane-bound vesicles containing hydrolytic enzymes.

- Function: Break down waste materials and cellular debris; involved in apoptosis.

Peroxisomes

- Structure: Small, membrane-bound organelles containing enzymes for oxidative reactions.

- Function: Break down fatty acids and detoxify harmful substances.

Chloroplasts (in plant cells)

- Structure: Double-membraned organelles with internal thylakoid membranes.

- Function: Site of photosynthesis; convert light energy into chemical energy.

Vacuoles (larger in plant cells)

- Structure: Membrane-bound sacs.

- Function: Storage of nutrients, waste products, and maintenance of turgor pressure in plant
cells.

3. Mechanisms of Compartmentalization

Membrane-bound Organelles

- Each organelle is surrounded by a lipid bilayer membrane that separates its internal
environment from the cytoplasm. This allows for specific conditions and environments
necessary for various biochemical processes.

Transport Vesicles
 66

- Small, membrane-bound vesicles transport materials between organelles. For example,

proteins synthesized in the ER are transported to the Golgi apparatus for further modification
and sorting.

Selective Permeability

- Organelle membranes are selectively permeable, allowing only specific molecules to enter or
leave. This selective transport is mediated by transport proteins and channels.

Signal Sequences and Receptors

- Proteins and other molecules often have specific signal sequences that direct them to their
correct compartment. Receptors on organelle membranes recognize these signals and facilitate
proper targeting and transport.

Compartment-specific Enzymes and pH

- Each compartment contains a unique set of enzymes suited to its specific functions.
Additionally, different compartments can maintain distinct pH levels, which is crucial for the
optimal activity of their resident enzymes.

4. Evolutionary Perspective

Endosymbiotic Theory

- Mitochondria and chloroplasts are believed to have originated from ancient symbiotic
relationships between primitive eukaryotic cells and prokaryotic cells. This theory is supported
by the fact that both organelles contain their own DNA and have double membranes.

Development of Internal Membranes

- The evolution of internal membranes likely provided early eukaryotic cells with a selective
advantage by allowing for more efficient organization and regulation of cellular processes.

5. Compartmentalization in Prokaryotes

While prokaryotic cells lack the complex compartmentalization seen in eukaryotes, they do
exhibit some degree of internal organization:

- Nucleoid: Region where the cell’s genetic material is located, though not membrane-bound.

- Inclusion Bodies: Serve as storage sites for nutrients and other substances.

- Microcompartments: Protein-based structures that compartmentalize specific metabolic

processes.
67
 68

Unit III

BIO-MEMBRANE TRANSPORT

Physiochemical of Plasma Membrane:-

Lipids

The plasma membrane contains about 20 to 79% lipids mainly of three types like phospholipids,
cholesterol and glycolipids. The phospholipids which make up between 55% and 75% of the
total lipid content, consists chiefly of lecithin and cephalin. The remainder Consists of
sphingolipids (with an amino group) and glycolipid conjugates with carbohydrates.
Phospholipids derived from glycerol are called phosphoglycerides. A phosphoglycerides is made
up of two fatty acid chains, a glycerol backbone and a phosphorylated alcohol. The outer layer

of phospholipids consists mainly of lecithin and sphingomyeline, while the inner layer is
composed mainly of phosphatidyl ethanolamine and phosphatidyl serine (both are
phosphoglycerides). The glycolipids (sugar containing lipids) are mainly in the outer half of the
bilayer.

A phospholipids cholesterol complex of cell membrane

Cholesterol is present in eukaryotes but not in prokaryotes. Plasma membrane of cells such as
erythrocyte, liver cells and myelinated nerve cells are rich in cholesterol. surrounding some
nerve axons) is composed of about 80% lipids and 20% protein and Membrane lipids are
amphipathic molecules. They contain both a hydrophobic and hydrophilic moiety. Hydrophilic
unit is also called the polar head groups, is represented by a circle and their hydrocarbon tails
are depicted by straight or wavy lines. Polar head groups have affinity for water, whereas their
 69

hydrocarbons tails avoid water. This can be accomplished by forming a micelle, in which polar
head groups are on the surface and hydrocarbon tails are directed inside.

A phospholipids molecule

Another arrangement of lipid molecule in a membrane is a bimolecular sheet, which is also called
a lipid bilayer. Phospholipids and glycolipids are key membrane constituents of bimolecular
sheets. Hydrophobic interactions are the major driving force for the formation of lipid bilayer.
The lipid bilayer of the membrane is interrupted only by the proteins that traverse it. This
bilayer consists primarily of:

Neutral Phospholipids and Cholesterol: These include phosphatidylenoline, lecithin cerebroside,

and sphingomyeline and phosphatidyl ethanolamine. They are without any electric charge at
neutral pH and are closely packed in the bilayer along with cholesterol.

Acidic Phospholipids: These constitute about 5% to 20% fractions of the total phospholipids of
plasma membrane. They are negatively charged and are associated with proteins by way of
lipid-protein interactions. Common examples are phosphatidyl inositol, Cardiolipin.
phosphatidylserine, sulpholipids, phosphatidyl glycerol and In plasma membrane, lipid fractions
form permeability barrier and structural framework

Proteins:-

Proteins are the main component of plasma membrane. Myelin sheath (membrane
surrounding some nerve axons) is composed of about 80% lipids and 20% protein and
presence of lipid makes myelin an excellent insulator. Eukaryotes membrane which serves
primarily as permeability barriers possesses about 50% proteins and 50% lipid. Plasma
membrane that are actively involved in energy transfer, such as inner membrane of
mitochondria, chloroplasts and membranes of aerobic prokaryotes have large amounts of
 70

proteins i.e. about 75%. They not only provide mechanical support but also act as carriers or
channels, serving for transport. In addition numerous enzymes, antigens and various kinds of
receptor molecules are present in plasma membranes. Membrane proteins are classified as
integral (intrinsic) or peripheral (extrinsic) according to the degree of their association with the
membrane (Singer, 1971).

Peripheral Proteins: They are also called extrinsic proteins associated with membrane surface.
These can be separated by addition of salts, soluble in aqueous solutions and usually free of
lipids. They are bound to the surface by electrostatic and hydrogen bond interactions. They form
outer and inner layers of the lipid bilayer of plasma membrane. Common examples are
cytochrome-C found in mitochondria, acetyl cholinesterase in electroplax membrane and
spectrin found in erythrocytes.

Integral or Intrinsic Proteins: These proteins penetrate the lipid layer wholly or partially and
represent more than 70% of the two protein types. Their polar ends protrude from the
membrane surface while non-polar regions are embedded in the interior of the membrane.
Usually they are insoluble in water solutions and can be separate them from the membrane by
detergents or organic solvents. The majorintegral proteins span the thickness of the membrane
and have a small amount of carbohydrates on the pole at the outer surface. This protein appears
to be involved in the diffusion of anions across the membrane. Integral proteins may be attached
to the oligosaccharides to form glycoprotein or to phospholipid to form lipoproteins or
proteolipids. Common intrinsic proteins are rhodopsin found in retinal rod cells and cytochrome
oxidase found in mitochondrial membranes.

Every protein in the cell membrane is distributed asymmetrically with respect to the lipid
bilayer.

Enzymes:-

About 30 enzymes have been found in various membranes. Those most constantly found are 5'-
nucleotidase, Na+-K+ activated ATPase, alkaline phosphatase, adenylcyclase, RNAse and acid
phosphomonoestrase. Na+-K+ activated Mg+ ATPase plays an important rolein the ionic exchange
and may also act as carrier protein or permease across the plasma membrane. Some enzymes
have a preferential localization. For example, alkaline phosphatase and ATPase are more
abundant in bile capillaries, while disaccharides are present in microvilli of the intestine.
Enzymes are asymmetrically distributed, for example in the outer surface of erythrocytes there
are acetylcholinestrase, nicotinamide adenine dinucleotidase and Na+-K+ ATPase. In the inner
surface there is NADH-diaphorase, G3PD, adenylate cyclase, protein kinase and ATPase.

Carbohydrates
 71

The membranes of eukaryotic cells usually contain 2% to 10% carbohydrates in the form of
glycolipids and glycoproteins. Hexose, hexosamine, fucose and sialic acid are the commonest
carbohydrates found in the membrane. Plasma membranes of neuronal surface contain
gangliosides (Lapertina, 1967) and are probably involved in the ion transfers. The distribution
of oligosaccharides is also highly asymmetrical.

Salts and water:-

They are also present in cell membranes. Water in cell membranes forms parts ofmembrane
structure as it does in all cell constituents.

Lamella-model of plasma membrane (Danielli-Davson model)

Danielli-Davson model (1934) suggested that the plasma membrane consists of twolayers
of lipid molecules arranged radially with their hydrophobic hydrocarbon chains toward each
other and with their respective polar groups arranged outwardly and inwardly
throughout the entire double layer of lipid molecules. The polar ends of the lipid molecules
are associated with a monomolecular layer of polar globular protein molecule. The entire
structure thus consisted of double layer of lipid molecule sandwiched between two
continuous layers of protein. The lipid molecules are set at right angles to the surface and are so
arranged in two layers that their non-polar hydrophobic fatty acid tails face each other and their
polar hydrophilic phosphate heads face the protein layer. The proteins involved were thought
to be globular. Moreover, lamellar theory assumed the cell membrane to be a stable structure
with little functional specificity and variability.
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A schematic diagram of Davson-Danielli model of membrane structure

A modification of original Danielli-Davson model, showing pores lined by polar proteinmolecules

extending through the lipid bilayer

Miceller model of plasma Membrane:-

According to the view of Hiller and Hoffman (1953), plasma membrane consists of amosaic of
globular subunits or micelles. If fatty acid molecules are completely surrounded by water, they
may form aggregate called micelles in which the hydrophobic regions of fattyacid molecules
are oriented toward the interior of the micelle away from the aqueous phase and their
hydrophilic groups are at the surface in contact with the surrounding water. Micelles may be in
the form of small spheres of bimolecular layers. These micelles are closely packed together
having a central core of lipid molecules and hydrophilic shell of polar groups. Each lipid micelle
measures 40Å to 70Å in diameter. Protein component of the plasma membrane forms a
monolayer on either side of the lipid micelles and is represented by globular type. The spaces
between the globular micelles are thought to represent water filled pores which measures about
4Å in diameter. These pores are bounded partly by the polar groups of micelles and partly by the
polar groups of associated protein molecules.

Plasma membrane based on Miceller theory (diagrammatic)

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Fluid Mosaic Model of plasma membrane:

It was proposed by Singer and Nicholson (1972). The lipids are thought to be arranged primarily
in a bilayer in which proteins are embedded to varying degrees. Singer classifies membrane
proteins as peripheral or integral. The proteins varied in size and dissolved to varying
degrees in the lipid matrix are able to diffuse laterally in the plane of membrane, and the entire
structure is hence dynamic. In this model, lipid molecules may exhibit intra molecular movement

or may rotate about their axis or may display flip-flop movement including transfer from one
side of bilayer to the other.

Plasma membrane based upon Fluid-mosaic model

The lipids, glycoprotein and many of the intrinsic proteins of the membranes are amphipathic
molecules. These amphipathic molecules constitute liquid crystalline aggregates in which the
polar groups are directed toward the water phase and the non-polar groups are situated inside
the bilayer. The lipid bilayer forms the structural matrix which serves as the permeability
barrier of the membrane. In membranes with high lipid content, lipid bilayer is extensive and
interrupted only occasionally by protein molecules, whereas in membranes with high protein
content, the extent of lipid bilayer is reduced. Thus, fluid mosaic model may describe the
chemical composition of the molecular organization and ultrastructure of plasma membranes.
This arrangement allows various enzymes and antigenic glycoprotein to have their active sites
exposed to the outer surface of the membrane. The fluidity of membrane also implies
that both the lipid and the protein have considerable freedom of movement within the
bilayer. The fluidity of the lipid depends on the degree of saturation of the hydrocarbon chains
and on the ambient temperature. A considerable proportion of the lipids in the membrane are
unsaturated, so that melting point of the bilayer is below body temperature.

Cell Respiration

Cell respiration takes place in mitochondria and so they are known as the 'power house' of the
cell. They bring about stepwise oxidation of food stuffs or "low-grade" fuel of the cell and transfer
the energy so released to the energy carrier ATP, the "high-grade" fuel of the cell. ATP is used to
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bring about the energy-requiring activities in the cells, namely, biosynthesis, active transport,
transmission of nerve impulse, muscle contraction, cell growth and division and
bioluminescence.

The plasma membrane is a selectively permeable barrier between the cell and the extracellular
environment. Its permeability properties ensure that essential molecules such as ions, glucose,
amino acids, and lipids readily enter the cell, metabolic intermediates remain in the cell, and
waste compounds leave the cell. In short, the selective permeability of the plasma membrane
allows the cell to maintain a constant internal environment.

The phospholipid bilayer, the basic structural unit of biomembranes, is essentially impermeable
to most watersoluble molecules, ions, and water itself. After describing the factors that influence
the permeability of lipid membranes, we briefly compare the three major classes of membrane
proteins that increase the permeability of biomembranes. We then examine operation of the
simplest type of transport protein to illustrate basic features of protein-mediated transport.
Finally, two common experimental systems used in studying the functional properties of
transport proteins are described

Few Molecules Cross Membranes by Passive Diffusion

Gases, such as O2 and CO2, and small, uncharged polar molecules, such as urea and ethanol, can
readily move by passive (simple) diffusion across an artificial membrane composed of pure
phospholipid or of phospholipid and cholesterol.

Such molecules also can diffuse across cellular membranes without the aid of transport proteins.
No metabolic energy is expended because movement is from a high to a low concentration of the
molecule, down its chemical concentration gradient.
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The 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.
All transport proteins are transmembrane proteins containing multiple membrane-spanning
segments that generally are helices. By forming a protein-lined pathway across the membrane,
transport proteins are thought to allow movement of hydrophilic substances without their
coming into contact with the hydrophobic interior of the membrane.

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.

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 (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.

Diffusion

Diffusion is defined as the net movement of a substance or molecules from a region of higher
concentration to a region of lower concentration. It is very useful in the separation of a variety
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of gases. Diffusion is impossible when the concentration gradients of both regions are the same.
For diffusion, we will have to create differentiation between the concentration gradients of two
cells. Simple diffusion occurs when electrochemical potentials on both sides of a permeable
barrier vary. Some factors affecting diffusion are the size of the molecule, concentration gradient,
temperature, and state of matter. Diffusion promotes cellular respiration and distributes
nutrients among cells and others.

Simple Diffusion

In simple diffusion, a substance moves from a higher concentration to a lower concentration. It

is a means of passive transport. Passive transport means that energy is not needed for the
movement of the molecules.

In this type of diffusion, very small molecules can move through gaps. It takes place between the
various phospholipid molecules in the cell membranes. The speed of simple diffusion is very low
and does not require any energy.

Inhibitor molecules do not hinder simple diffusion. In this type of diffusion, the movement of
particles takes place in the direction of a concentration gradient. Water and oxygen are examples
of molecules that use simple diffusion.

Examples of simple diffusion

 Steroid hormones can move freely across different membranes down their
concentration gradient. So, this is an excellent example of simple diffusion.

 At the time of food digestion, oxygen is transferred into the blood from the lungs. Also,
oxygen is transferred into the muscles from the blood cells.

 In the body of pregnant women, food and oxygen travel from the mother’s body into the
fetus.

 Bacteria have single-celled microorganisms. Bacteria deliver water, oxygen, and

nutrients to the cytoplasm. No specialised organelles in bacteria perform this type of
activity.

 Dialysis is an artificial process that partially replaces renal function. Dialysis is based on
the principle of diffusion of solutes and ultrafiltration of fluid across a semipermeable
membrane.
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Diffusion of electrolytes

 The principle for the diffusion of charged species is driven by an additional force along
with the concentration gradient.

 Charged solutes are subject to electrical forces when electrostatic potential gradients are
present.

 Accordingly, the driving force for electrolyte transport is the gradient of

the electrochemical potential rather than that of the chemical potential.

 Since any electrolyte solution must contain at least one anion and one cation, there are
always at least two solute species which results in multiple fluxes.

Factors affecting simple diffusion

1. Concentration gradient

 The concentration gradient across a biological membrane is the driving force for the
diffusion of a nonelectrolyte.

 Therefore, the higher the concentration difference across the membrane, the higher will
be the rate of diffusion.

 As the distribution of molecules across the membrane moves towards uniformity, the
rate of diffusion decreases.

 Once equilibrium is maintained across the membrane, the process of diffusion ceases.

2. Mass/Size of the solute molecules

 The size of the molecules also affects the rate of diffusion across a biological membrane.

 If the size of the molecules is large, it will be more difficult for it to move across the
membrane, which, in turn, slows the rate of diffusion of the molecule.
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 Thus, the rate of diffusion is higher for smaller molecules and slower for larger
molecules.

3. Temperature

 The temperature of the system also affects the process of simple diffusion.

 With the increase in temperature, the energy of the molecules also increases.

 Molecules with higher energy can move faster across the membrane while particles with
lower energy move slower.

4. Solubility

 The solubility of molecules in a medium also affects the rate of diffusion of the particles.

 Molecules which are lipid-soluble can move quickly across the lipid layer like the plasma
membrane.

 Similarly, polar and non-polar molecules also move with a different rate depending on
the nature of the biological membrane.

5. Solvent density

 With the increase in solvent density, the rate of diffusion decreases.

 More dense a solvent, more difficult it will be for the solute to move around.

 Solvent density plays an essential role in the movement of solute in the cytoplasm of the
cell.

 An increase in the density of the cytoplasm slows down the movement of the molecules
and gases, and the reverse is true for less dense cytoplasm.

6. Surface area and thickness of the biological membrane

 The rate of diffusion increase with the increase in the surface area of the membrane.

 The increase in surface area increases the permeability or mobility of the molecules as
mobility is one of the factors responsible for the flux.

 Similarly, the rate of diffusion is also reduced by the increasing thickness of

the membrane

Facilitated diffusion is the process of biological transport in which specific structural

components of biological membranes interact with particular solutes or classes of solutes,
markedly increasing the rates at which they can cross the membrane.
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 It is a passive-mediated transport in which particles or substances are transported

across a biological membrane from a region with a higher concentration of an area of
lower concentration facilitated by some transport protein.

 As the movement of substances occurs in the direction of the concentration gradient

(from higher to lower), no chemical energy or ATP is required.

 However, the substances which are transported via facilitated diffusion would not
otherwise move easily or quickly across the membrane.

 Similarly, the membrane components responsible for facilitated diffusion are called
transport mediators.

 The lipid bilayer of a plasma membrane does not allow the transport of all molecules
with the same ease.

 Since the membrane is hydrophobic, it doesn’t allow the movement of hydrophilic as

well as some highly polar m molecules.

 Few of the hydrophilic molecules, along with smaller hydrophilic molecules, can quickly
move across the membrane based on the concentration gradient.

 However, the larger non-polar molecules require aid from transport mediators like the
membrane carriers and channels.

 The movement across the membrane can be made through one of the two mechanisms;
one involving the carrier proteins and the other involving the channel proteins.
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 In the case of channel proteins, the transmembrane proteins present in the membrane
act like a channel (pore) in the membrane, which allows the transport of the molecules.

 These channels extend across the plasma membrane, connecting the external
environment to the cytosol or reaching across the biological membranes of different
cellular organelles.

 Molecules like-charged ions are transported via the transmembrane channels formed by
protein complexes.

 In the case of carrier proteins, transporters, or carrier proteins embedded in the

biological membrane are utilized.

 These proteins have a specific affinity towards some molecules on the extracellular
matrix.

 The carrier proteins bind to the molecules which results in some conformational changes
in the molecules, facilitating the movement across the membrane into the cytosol.

 This mechanism of facilitated diffusion is employed for larger molecules like enzymes.

Channel Proteins

 Channel proteins are integral proteins present in the biological membrane that allow the
transport of molecules across the membrane by forming a channel.

 The species that pass through channels, also called the transmembrane proteins as they
span across membranes, are almost always ions.

 Many channels are very selective, passing some ions readily while being substantially
impermeable to others.

 Based on structural models, channel diameters are thought to be no more than 4 – 5 Å,

similar to the widths of the common biological ions.

 Similarly, channels may pass cations readily but not anions or may exhibit very different
permeabilities to two ionic species having the same charge, thus increasing the
selectivity and specificity of the diffusion.

 These channels have hydrophilic domains exposed to both the extracellular and
intracellular matrix.

 Additionally, they have a hydrophilic core that provides a hydrated opening through the
membrane layers.
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 Aquaporins are the channels proteins that transport water across the plasma membrane
at a very high rate.

 The selectivity properties of channels arise from interactions between the ions and the
mouth or walls of the pores.

 The passage of ions through these channels allows the movement while avoiding the
non-polar central layer of the plasma membrane.

 Channel proteins are usually gated and allow the closing or opening of the channels
based on some signals.

 These signals can either be an electrical signal or simply binding of a molecule.

Carrier Protein

 Carrier proteins are another group of proteins involved in facilitated diffusion present
in the membranes.

 Carrier proteins, as the name suggests, carry molecules across the membrane.

 These proteins bind to specific regions in the molecules causing conformational changes
and then move the bound molecule to the interior of the cell depending on the
concentration gradient.

 Carrier proteins are bulky, and it is unlikely that they transport solute by diffusing from
one face of the membrane to the other.

 Therefore, in most of the models, the carrier accomplishes its task by a conformational
change.

 The mechanism of the conformational change is not entirely understood, but it is

assumed that the hydrogen bonds are affected, which results in a change in the shape of
the molecule.

 Binding sites on carriers are very selective. For example, sugar carriers distinguish
between d- and I-sugars.

 The configuration of the binding site, or the charge distribution at the site, should match
that of a distinctive portion of the desired substrate.

 This selectivity adds to the selectivity of the plasma membrane.

 The rate at which a particular substrate is carried across the membrane can be
influenced by other solutes in the system.
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 At a point where all the carrier proteins are bound to their ligands, they become
saturated, and the transport rates become the maximum.

 Carrier proteins are also used in active transport for the movement of molecules with
the expense of energy.

Examples of facilitated transport

1. Glucose and amino acid Transport

 The transport of glucose and amino acid from the bloodstream into the cell is an example
of facilitated diffusion.

 In the small intestine, these molecules are taken in via active transport and then are
released into the bloodstream.

 Because glucose and amino acid are larger molecules, they require carrier proteins
called glucose transporters or amino acid permeases, respectively for their transport
from the bloodstream into the cell.

2. Gas Transport

 The transport of oxygen in the blood and muscles is another example of facilitated
diffusion.

 In blood, hemoglobin is the carrier protein whereas in muscles, the carrier protein in the
myoglobin.

 The diffusion of blood occurs as a result of higher pressure on one side of the membrane
and a lower one on the other side.

 A similar mechanism is involved in the transport of carbon dioxide and carbon

monoxide.

3. Ion Transport

 Ions are polar molecules and thus cannot move across the membranes with similar
charges.

 These ions are transported via transmembrane proteins which are called the ion
channels.

 These channels are specific for specific ions like potassium, sodium, and calcium.

 These channels are highly specific and allow fast transport rates without using any
chemical energy.
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The membranes apply two modes of transport for molecular movement, this includes:

Passive Transport – This transport mechanism involves the movement of substances across
the membrane down the concentration gradient from high to low without employing any energy
expenditure.

Examples include:

 The diffusion of neurotransmitters like acetylcholine across synaptical junctions in

neurons.

 Diffusion of gases in the lungs across alveolar membranes.

 Movement of water molecules by osmosis.

Active Transport – A transport mechanism that involves the movement of molecules across the
membrane against the concentration gradient, meaning the transport of substances from low to
high concentration. This movement is achieved by the energy expenditure from ATP (adenosine
triphosphate).

Examples include:

 Movement of Ca2+ ions out of the cardiac muscle cells.

 Transportation of glucose molecules across membranes.

 Movement of amino acids in the human gut across the intestinal lining.

Lipid Bilayer and Membrane Proteins

 Most biological membranes are composed of a phospholipid bilayer, with the

hydrophilic side exposed outwards and the hydrophobic side facing inwards, and
transmembrane proteins spanning the bilayer.

 The integrated proteins present in the membrane form the transport channel, pumps,
and carriers that are required for the translocation of ions and macromolecules across
biological membranes.

 Channel proteins form pores in the membrane to allow free passage of inorganic ions
like Na+, Cl-, K+, H+, etc, and molecules of appropriate size.

 The channel proteins are strictly regulated by extracellular signals to control the
movement of ions and solutes across the membrane.

 Carrier proteins act like an enzyme that selectively binds to and transports specific small
molecules such as glucose to facilitate the translocation across the membrane.
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 The composition of the phospholipid-to-protein ratio varies depending on the type of

cell it is present in. For example, the mitochondrial inner membrane has 75% protein
suggesting the presence of protein complexes involved in oxidative phosphorylation and
the electron transport system.

Active Transport

Active transport is the energy-driven transportation of ions, small molecules, and solutes
across the biological membrane against an electrochemical gradient (for ions) or
concentration gradient i.e., from lower to higher concentration.

Since this mode of cellular transportation requires energy, a significant amount of cellular
energy is spent on regulating the homeostasis of molecules and ions in a cell. Active transport is
further divided into two types – Primary and Secondary active transport.

Types

Classification of Active Transport is based on how energy is employed to bring about the
movement of substances. Primary active transport is seen to use ATP as an energy source to
translocate solutes against a concentration gradient, whereas in Secondary active transport, an
electrochemical gradient is applied.

Primary types

This category of active transport directly employs the use of metabolic energy to translocate
substances across the membrane, hence also called as ‘Direct Active Transport.’ The
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transportation of molecules uses energy released from ATP, where Adenosine triphosphate
(ATP) is broken down to ADP (adenosine diphosphate). This transport mode is used to move
metal ions like K+, Na+, Cl-, H+, Mg2+, and Ca2+. The enzymes utilized for the primary active
transport of ions are ATPases that facilitate the movement of charged ions across ion channels
or pumps.

Types of Primary Active Transport

P-Type ATPases

They are also referred to as E1-E2 (enzyme1-enzyme2) ATPases due to their ability to
interchange between two conformations.

 In humans, the pumps with P-type ATPases show diverse roles in nerve impulses,
absorption of nutrients and solutes in the intestine and kidney, and in relaxation of
muscles.

 Eukaryotes and bacteria have ion channels with P-type ATPases.

 The ‘P-type’ refers to the autophosphorylation among components present in the pumps,
such as ATP and aspartate, present in the enzyme structural composition.

 The enzyme structure typically contains four significant functional domains to carry out
the transportation process, which include

1. P-domain (Phosphorylation) containing amino acids like aspartate.

2. N-domain – Used for nucleotide binding.

 86

3. A-domain – Actuator domain which contains a phosphatase domain for

dephosphorylation of the phosphorylated element.

4. R-domain – Regulatory domain.

Examples of P-type ATPases involved in ions pumps include Na+-K+ (sodium-potassium ) ion
channel, Calcium ATPase (Ca2+ pump), Hydrogen ATPase (H+ pump), and H+/K+ ATPase in
proton-potassium pump, in fungi, bacteria, humans, and plants.

Mechanism of Sodium-Potassium (Na+/K+ ATPase) Pump

This pump employs a direct utilization of ATP to bring about the conformational changes to the
protein and allow passage of three Na+ ions out of cells while bringing two K+ ions into the cell.

 The transmembrane carrier protein is opened towards the cell interior with a high
affinity for sodium ions.

 The binding of sodium ions to the carrier protein induces phosphorylation of the
transmembrane protein by ATP hydrolysis.

 The phosphorylation of protein modifies the structural conformation making it open to

the cell exterior, leading to a lower affinity for sodium ions, hence releasing the three
bound sodium to the extracellular space.

 The outward-facing carrier protein conformation shows a higher affinity for potassium
ions.

 As the potassium ions bind to the carrier protein, it releases the phosphate group
attached to the protein.

 The detachment of the phosphate group from the protein causes it to change to its
original conformation facing to the cell interior.

 The change in conformation loses the affinity for potassium ions, hence releasing it into
the cell, and again sodium ions can bind to the protein, and the cycle repeats
continuously.

 A cardiac glycoside called ‘Ouabain’ blocks the influx of potassium ions into the cell by
binding to the protein surface and inhibiting the dephosphorylation of carrier protein.
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F-ATPase

They are mainly found in the mitochondrial inner membranes and chloroplast’s thylakoid
membrane.

 They are sometimes also referred to as ATP phosphohydrolase that transports H+ ions,
or as ATP synthase.

 The enzyme uses rotating machinery to pump Na+ or H+ ions which are powered by
energy derived from ATP hydrolysis.

 F-type ATPase contains two main domains – F1 and F0.

 F1 Domain – It is a Hydrophilic catalytic globular domain made of an asymmetric

hexameric ring and a central stalk at the center of the ring. This domain contains catalytic
sites for ATP hydrolysis and synthesis.

 F0 Domain – Responsible for the translocation of ions across a membrane. It is

hydrophobic in nature and is embedded in the membrane.

 The energy obtained from the movement of protons across the membrane is used to
drive ATP synthesis, which helps in releasing newly formed ATP from F-ATPase’s active
site.

 ATP hydrolysis is done at conditions of a lower driving force. Hence ATPase functions as
ATP synthase that generates a transmembrane electrochemical gradient.

V-ATPase
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The V denotes vacuolar ATPases, which are proton-translocating pumps present in the
tonoplast of cells. They are responsible for making the organelle more acidic than the
cytoplasm around them.

 This enzyme employs the energy obtained from ATP hydrolysis to drive the transport of
protons across biological membranes.

 It is a multimeric protein that uses rotatory machinery to allow the passage of ions and
has two distinct domains – V1 and V0.

 V1 Domain – Contains catalytic site for ATP hydrolysis and comprises eight subunits.

 V0 Domain – This domain is responsible for the movement of protons across a

membrane.

 They are considered an important molecular switch that toggles between anabolic and
catabolic metabolism, and any disruption to their structure can lead to diseases like
cancer.

ATP Binding Cassette (ABC) Transporters

They are significantly found in chloroplasts, mitochondria, and plasma membranes and are seen
to play a vital role in detoxification, phytohormone transport, and in pathogen response. They
utilize the energy released from the hydrolysis of ATP to allow the passage of solutes across a
membrane.

 They have four significant domains, two of which are transmembrane integral domains
spanning the membrane six times, and the other two domains are responsible for ATP
hydrolysis.

 They are actively seen to export antimicrobial metabolites and volatile compounds such
as benzene, 1,3-butadiene, etc.

 Approximately 48 genes are identified in humans, and most of these genes are part of
diseases like Dubin-Johnson Syndrome, Cystic Fibrosis, Ataxia, Anemia,
adrenoleukodystrophy, and many more.

 They participate in processes like drug/antibiotic resistance, signal transduction,

antigen presentation, bacterial pathogenesis, etc.

Secondary Active Transport

Also called as Coupled Transport or Cotransport, as there is a movement of ions along with
large molecules like glucose. First, the ions are pumped down the electrochemical gradient
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but against a concentration gradient, as a result of which energy is released. Then the solute is
translocated across a membrane down the concentration gradient.

 The functions performed by this mode of transport include the generation of an

electrochemical gradient due to the movement of ions at the expense of energy across a
membrane.

 The ion gradient difference leads to a movement of solute in either the opposite
direction, referred to as Antiport, or in the same direction, referred to as Symport.

 In yeast and bacteria, hydrogen (H+) ions are the commonly cotransported ion, whereas,
in humans, sodium (Na+) ions are widely used for coupled transportation.

Secondary Active Transport Types

There are basically two modes of secondary active transport based on the direction of solute
along with ions across a membrane: Antiport (opposite direction) and Symport (same
direction). The level of effective movement of substances can be measured by the concentration
capacity of the transporters to translocate ions/solutes in the process per cycle. The higher the
ion/substrate coupling ratio, the higher the concentration capacity of the transporter. For
instance, the coupling of sodium (Na+) ions to glucose results in the movement of two sodium
ions to one glucose molecule per transport cycle with a ratio of 2:1.

Antiport

Also referred to as Counter transport or Exchanger as solutes and ions are allowed to move
in the opposite direction. Examples include the Na+/Ca2+ transporter via calcium ATPase, Cl-
/HCO3- transporter, and Na+/H+ transporter.

 Na+/Ca2+ Antiporter – This antiporter is necessary to regulate low calcium

concentration in the cardiac muscle cells. This exchanger allows three Na+ ions down
the electrochemical gradient and one Ca2+ ions against the electrochemical gradient
from low to high solute concentration with a stoichiometric coupling ratio of antiporter
3:1.

 Cl-/HCO3- Antiporter – Results in electroneutral exchange as one Cl- ion down its
electrochemical gradient per bicarbonate (HCO3-) ion against the electrochemical
gradient with a stoichiometric coupling ratio of antiporter 1:1.

 Na+/H+ Antiporter – Electroneutral mechanism and very useful in maintaining the

cytoplasmic pH as one Na+ ion is transported down its electrochemical gradient per H+
ion against its gradient with a stoichiometric coupling ratio of antiporter 1:1.
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Symporter

The solutes and ions are transported in the same direction, with one substance being
translocated down its concentration gradient while the other substance is moved against its
gradient from low to high solute concentration. Examples include glucose symporter (SGLT1),
GABA symporter (GAT), oligopeptide symporter (PepT), and more.

 Glucose symporter (SGLT1) – It can be found in the cells of the intestinal epithelium,
nephrons of the kidney, brain, and in heart. Two molecules of Na+ ions are imported
down its concentration gradient into the cell per glucose (or galactose) molecule against
the gradient.

 GAT Symporter – Responsible for regulating the concentration of GABA (gamma-

aminobutyric acid), an inhibitory neurotransmitter in the synaptic cleft of the nervous
system. The symporter is coupled with Na+ or Cl- ions for solute movement across a
membrane.

 Oligopeptide symporter (PepT) – Serves as a crucial entry route for drugs like beta-
lactam antibiotics and helps in the reabsorption of nitrogen in the intestine and kidney.
In this symporter, one H+ ion is transported from high to low concentration per
dipeptide or tripeptide molecule against its concentration gradient.
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ATP-Powered Pumps and the Intracellular Ionic Environment

The transport ions and various small molecules against their concentration gradients. All ATP-
powered pumps are transmembrane proteins with one or more binding sites for ATP located on
the cytosolic face of the membrane. Although these proteins commonly are called ATPases, they
normally do not hydrolyze ATP into ADP and Pi unless ions or other molecules are
simultaneously transported. Because of this tight coupling between ATP hydrolysis and
transport, the energy stored in the phosphoanhydride bond is not dissipated but rather used to
move ions or other molecules uphill against an electrochemical gradient.

Different Classes of Pumps Exhibit Characteristic Structural and Functional Properties

The general structures of the four classes of ATP-powered pumps are depicted in Figure 7-6,
with specific examples in each class listed below. Note that 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 are thought to move through the phosphorylated subunit. The
sequence around the phosphorylated residue is homologous in different pumps. This class
includes the Na/K ATPase in the plasma membrane, which main tains the low cytosolic Na and
high cytosolic K concentrations typical of animal cells. Certain Ca2 ATPases pump Ca2 ions out
of the cytosol into the external medium; others pump Ca2 from the cytosol into the endoplasmic
reticulum or into the specialized ER called the sarcoplasmic reticulum, which is found in muscle
cells. Another member of the P class, found in acid-secreting cells of the mammalian stomach,
transports protons (Hions) out of and Kions into the cell. The Hpump that generates and
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maintains the membrane electric potential in plant, fungal, and bacterial cells also belongs to
this class.

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 known V and F pumps transport only protons, in a
process that does not involve a phosphoprotein intermediate. V-class pumps generally function
to maintain the low pH of plant vacuoles and of lysosomes and other acidic vesicles in animal
cells by pumping protons from the cytosolic to the exoplasmic face of the membrane against a
proton electrochemical gradient. F-class pumps are found in bacterial plasma membranes and
in mitochondria and chloroplasts. In contrast to V pumps, they 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.

Na+/K+ ATPase Maintains the Intracellular Na+/K+ Concentrations in Animal Cells

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 subunits to fold properly in the
endoplasmic reticulum but apparently is not involved directly in ion pumping. The amino acid
sequence and predicted secondary structure of the catalytic subunit are very similar to those of
the muscle SR Ca2+ ATPase In particular, the Na/K ATPase has a stalk on the cytosolic face that
links domains containing the ATP-binding site and the phosphorylated aspartate to the
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membrane-embedded domain. The overall transport process moves three Na ions out of and
two K ions into the cell per ATP molecule hydrolyzed.

The mechanism of action of the Na/K ATPase, the muscle calcium pump, except that ions are
pumped in both directions across the membrane. In its E1 conformation, the Na/K ATPase has
three high-affinity Na binding sites and two low-affinity K binding sites accessible to the
cytosolic surface of the protein. The Km for binding of Na to these cytosolic sites is 0.6 mM, a
value considerably lower than the intracellular Na concentration of ≈12 mM; as a result, Na ions
normally will fully occupy these sites. Conversely, the affinity of the cytosolic K binding sites is
low enough that K ions, transported inward through the protein, dissociate from E1 into the
cytosol despite the high intracellular Kconcentration. During the E1 n E2 transition, the three
bound Na ions become accessible to the exoplasmic face, and simultaneously the affinity of the
three Na binding sites becomes reduced. The three Na ions, transported outward through the
protein and now bound to the low-affinity Na sites exposed to the exoplasmic face, dissociate
one at a time into the extracellular medium despite the high extracellular Na concentration.
Transition to the E2 conformation also generates two high-affinity K sites accessible to the
exoplasmic face. Because the Km for K binding to these sites (0.2 mM) is lower than the
extracellular K concentration (4 mM), these sites will fill with K ions. Similarly, during the E2 n
E1 transition, the two bound K ions are transported inward and then released into the cytosol.
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Only one of the two catalytic subunits of this P-class pump is depicted. It is not known whether
just one or both subunits in a single ATPase molecule transport ions. Ion pumping by the Na/K
ATPase involves phosphorylation, dephosphorylation, and conformational changes similar to
those in the muscle Ca2 ATPase. In this case, hydrolysis of the E2–P intermediate powers the E2
n E1 conformational change and concomitant transport of two ions (K inward. Na ions are
indicated by red circles; K ions, by purple squares; high-energy acyl phosphate bond, by ~P; low-
energy phosphoester bond, by –P

Transport by vesicle formation, endocytosis, exocytosis

Biological membranes are external boundaries of cells that define external boundaries and
regulate the exchange of essential components across these boundaries. The physical properties
of phospholipid bilayer such as flexible structure, selectively permeable and self sealing, allow
cells to change their shapes that is essential during cell division. Sealing properties allow fusion
of two membranes and become one as well as fission of single membrane results in two sealed
compartments without any leakage through cellular surfaces.

‘Exocytosis’ is export of materials from inside of the cell via vesicles using secretory pathway
which involves the fusion of the membranes of secretory vesicles with the plasma membrane. It
results in the release of the vesicle contents from the cell and also the delivery of vesicle
membrane proteins into the plasma membrane. Exocytosis is important in expulsion of waste
materials out of the cell and in the secretion of important cellular products such as digestive
enzymes, secretory proteins and hormones etc. During exocytosis, the Golgi complex packages
macromolecules such as proteins and polysaccharides into transport vesicles that further travel
to and fuse with the plasma membrane. This fusion with plasma membrane causes the vesicle to
secrete their contents out of the cell.

Endocytosis is a form of active transport in which a cell uptake materials from outside into the
cell with the help of an energy-using process (Figure 1). Endocytosis is in general the
counterpart of exocytosis and used by majority of cells because most chemical substances
important to them are large polar molecules that cannot pass through the hydrophobic plasma
or cell membrane by passive transport. Cells use three major types of endocytosis: phagocytosis,
pinocytosis, and receptor-mediated endocytosis.
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The first stage of vesicular transport is the production of membrane bound small transport
vesicles. Small vesicles formation requires deformation of the lipid bilayer by which a
gobletshaped invagination of the membrane appear that will eventually be pinched off to form
the vesicle. This process is termed as budding. The budding from parental membranes and
formation of new vesicle is driven by interactions and polymerization of soluble proteins.

Vesicle formation is an energy-dependent process and is mediated by proteins such as epsins,

which are required specifically for budding. A formed vesicle carries the proteins that were
present in that stretch of membrane as well as the molecules that has to be transported. At last
fusion of the vesicle occur with the membrane of target location where the soluble molecules
reaches its destination. The proteins that direct the targeting of the vesicle to the correct cellular
location also mediate fusion, and in some systems regulate the precise time at which fusion
occurs. Several vesicles have a coat of proteins surrounding their membrane and are therefore
called coated vesicles. The coat is acquired as the vesicle buds from the donor membrane and is
shed before the vesicle fuses with the target membrane. There are three types of coated vesicles
that help to localize biomolecules to particular compartments

 Clathrin-coated vesicles: Endocytic and secretory vesicles have Clathrin as the most
prominent protein in their coats and these Clathrin coated vesicles are responsible for
transportation of proteins from the plasma membrane (cell surface) into the cell and the
trans Golgi network to late endosomes (Figure).
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 COat Protein I (COPI) coated vesicles: The vesicles involved in transportation of proteins
in the retrograde direction between Golgi cisternae and from the cis- Golgi back to the
rough endoplasmic reticulum (Figure).

 COPII-coated vesicles: These vesicles are involved in trafficking from the rough
endoplasmic reticulum to the cis Golgi

EXOCYTOSIS: THE SECRETORY VESICULAR PATHWAYS

Exocytosis is the vesicular transport process by which molecules are released to outside of the
cell. Proteins are recruited to the plasma membrane as well as the release of secreted molecules
into the extracellular matrix occurs through exocytosis. In addition, higher eukaryotic cells
communicate with each other via a variety of intercellular signalling molecules (hormones,
cytokines and other small signalling molecules) that are released from secretory vesicles.
Moreover, exocytosis is responsible for the transmission of a signal from one cell to another
which occurs by the secretion of neurotransmitters into the synaptic cleft, in response to the
arrival of an action potential.

Exocytosis may be differentiated into ‘constitutive exocytosis’ and ‘regulated exocytosis’.

Constitutive exocytosis is carried out by nearly all cells to transfer molecules from the Golgi
apparatus to the plasma membrane of the cell. Constitutive exocytosis is not an unregulated
process; and the rate of release of the molecules to the cell surface depends on their rate of
production, which is regulated by transcription and translation. While, regulated exocytosis
occurs in response to specific biochemical signal which results the release of cytokines,
hormones, neurotransmitters and other small signalling molecules, such as histamine. Regulated
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secretion is typically controlled by external signals transduced via cell surface receptors.
Molecules in the trans Golgi network are sorted into secretory vesicles either for regulated
secretion if they contain appropriate signal sequences or for constitutive secretion if they do not.
For regulated secretion, proteins are first assembled in immature vesicles, and these vesicles
mature as secretory vesicles near the plasma membrane.

THE ENDOCYTIC PATHWAYS

The process in which cells internalise molecules from the outside into it’s cytosol is termed as
Endocytosis. Endocytic mechanisms control the lipid and protein composition of the plasma
membrane, thereby regulating how cells interact with their environments.

Endocytosis describes the de novo production of internal membranes from the plasma
membrane lipid bilayer. In so doing, plasma membrane lipids and integral proteins and
extracellular fluid become fully internalized into the cell. Endocytosis a key process in regulating
processes such as mitosis, antigen presentation, and cell migration. There are number of
endocytic mechanisms each of which involves unique proteins.

The endocytic pathway comprises two distinct kinds of endosome, early endosomes and late
endosomes. Material taken up by endocytosis passes from the early endosomes to the late
endosomes and from there may intersect with trafficking pathways from the Golgi apparatus, or
may be directed to lysosomes or to the Golgi. The exact pathway depends on the cell and the
material that has been internalised. Endocytic mechanisms are differentiated into three
subcategories: namely, macropinocytosis, phagocytosis, receptor-mediated endocytosis. In
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macropinocytosis small soluble molecules get the uptake in the form of vesicles (0.5–5 µm in
diameter), which usually occurs from highly ruffled regions of the plasma membrane. These
vesicles are filled with a large volume of extracellular fluid and molecules within it. No specificity
is required in filling of the vesicle. These vesicle travels into the cytosol and get fused with
endosomes and lysosomes in later stages.

Phagocytosis is an endocytic process in which cells bind and internalize larger insoluble
particles (0.75 µm in diameter) like cell debris, micro-organisms and apoptotic cells. Receptor-
mediated endocytosis is an endocytic uptake of specific molecule from the external medium
using a receptor which generally present on the membrane surface and have specificity for the
internalized molecule. In general, receptor-mediated endocytosis may occur by two different
types that are ‘clathrin-mediated’ and ‘caveolae’ endocytosis. Clathrin-mediated endocytosis
(CME) is mediated by the production of small (approx. 100 nm in diameter) vesicles that contain
a coat made up of the cytosolic protein ‘clathrin’. In this process cargo recruited into developing
clathrin coated pits (CCPs) and result in the formation of clathrin-coated vesicles (CCVs). Coated
pits can concentrate large extracellular molecules that have different receptors responsible for
the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth
factors, antibodies and many others. Clathrin consists of a heavy chain and a light chain and
successively assemble into a polyhedral, cage-like coat on the surface of the coated pit. The
clathrin coat is made of sub-assemblies, each consisting of a threepronged protein complex, a
triskelion, each leg of which is made of one heavy and one light chain (Figure 5a). The triskelion
forms a lattice-like network of hexagons and pentagons (Figure 5b), which attaches to the
membrane via an adaptor protein (AP) complex. Adaptor proteins bind both to clathrin and to
integral membrane proteins which are destined to be internalized and stimulate its assembly.
Much more importantly, by binding to the molecules in the membrane of the vesicle, adaptor
proteins appear to be responsible for recognizing the appropriate cargo molecules.
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Unit IV

CELL CYCLE

A multicellular organism starts its life as a single cell and it undergoes repeated division, thus,
the growth and development of every living organism depends on the growth and multiplication
of its cells. The cell increases in size due to growth and it is the characteristic feature of all the
living organisms. After the cell attains maximum growth, it begins to divide. The vegetative
growth of an organism takes place by an increase in the number of cells through cell divisions
which follows the geometrical progression. The cell division is a continuous and dynamic
process and it involves the following three stages:

1. DNA or genome replication

2. Nuclear division or karyokinesis

3. Cytoplasmic division or cytokinesis

The cell division is of two types on the basis of number of genomes present in the daughter cells
in comparison to the dividing parent cell — mitosis and meiosis.

1. Mitosis- The term mitosis was coined by W. Flemming in 1882. The multiplicationof a
body cell into two daughter cells of equal size and containing the same number of
chromosomes as in the parent cell is called mitosis or somatic division.

2. Meiosis- The term meiosis was first coined by J. B. Farmer (1905) with J. E. Moore.
Meiosis occurs only in gonads (in germ mother cells) during the formation of gametes like
sperm and ovum. Meiosis is a process by means of which double number or 2N or diploid
chromosomes is reduced to its half number or N or haploid. It is also called reduction
process. Cell Cycle Stages, Mitosis & Cytokinesis

Cell Cycle:-

Every cell having the capacity to divide passes through a regular cycle of changes known as
cell cycle. A cell starts its cycle in diploid condition.

Phases of cell cycle:-

Cell cycle consists of two stages: A long un-dividing stage called interphase or I-phase and
a short dividing stage called mitotic or M-phase
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Cell Cycle checkpoints

1. Interphase- The time between the end of telophase and the beginning of the next M- phase
is called the interphase. It is a long stage that lasts for 10 to 30 hours. During this phase
the cell grows by synthesizing biological molecules such as lipids, proteins,carbohydrates,
nucleic acids.

Interphase is further divided into three sub phases or periods: first gap or G1 phase, synthetic
or S phase and second gap or G2 phase.

(i) G1 phase- The gap between previous mitosis and beginning of DNA synthesis is
represented by G1 phase. In this stage initial growth of a newly formed cell takes place.
Various biological molecules (carbohydrates, proteins, lipids, including some non-
histones, RNAs) are synthesized in this phase. Normal metabolism is carried out for the
preparation for DNA

replication that is to take place next to it. DNA synthesis does not occur in thisphase.

(ii) S Phase- During this phase duplication of each chromosome take place by replication
of new DNA molecule on the template of the existing DNA.Synthesis of histone proteins
and their mRNA, some non-histone proteins and formation of new nucleosome also occur
in S-phase only. In most of the eukaryotes the S-phase lasts for 6 to 8 hours.

(iii) G2 Phase- G2 phase is the gap between DNA synthesis and nuclear division. RNA
transcription and protein synthesis continues during this phase. Further growth of the cell
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and preparation for its division also takes place in this stage. During this stage the
cytoplasmic organelles such as centrioles, mitochondria and Golgi apparatus are doubled,
proteins for spindle and asters are synthesized and active metabolism stores energy for
the next mitosis. The G2 phase in most cells lasts for 2 to 5 hours.

2. Mitotic Phase- Interphase is followed by mitotic phase. During mitotic phase the already
duplicated chromosomes are equally distributed to the daughter cells which contain
exactly the same hereditary information as the parent cell. Though, the other cell
components (organelles and molecules) are also divided approximately equally between
the daughter cells, but not as precisely as the DNA. After the mitosis is over, the daughter
cells enter the G1 phase of the next cell cycle.

During mitosis many structural and physiological changes take place in the cell, as thechromatin
of the nucleus is packed into visible chromosomes, which are set free by breakdown of
nuclear envelope. An extensive reorganization of the membranous components and
cytoskeletal elements takes place. Endoplasmic reticulum and Golgi apparatus break
down into small vesicles and stops the protein movement. Microtubules dissociate into
tubulin dimers and are assembled into the spindle which occupies most of the cell and
helps in the distribution of chromosomes into the daughter cells. Actin filaments get
reorganized and form a contractile ring for the cytoplasmic division.

Control of Cell Cycle:-

1. Nucleo-cytoplasmic Ratio- In 1910, Hertwig proposed that the cell division starts when
the ratio between the volume of the nucleus and the volume of the cytoplasm is
upset. As the cell grows, the synthesis of proteins, nucleic acids, lipids and other cellular
components takes place. During synthesis of these molecules, the back and forth
movements of materials through the nuclear and the cell membranes occurs. With the
growth of the cell, its volume increases more than the surface of the nucleus and the cell,
and at a critical point, the surface of the nucleus becomeinadequate for the exchange of
materials between the nucleus and the cytoplasm required for further growth. The cell
divides at this stage and regains the optimum and efficient nucleo-cytoplasmic ratio that
allows the growth. Although the cell division usually occurs after a cell has grown to a
certain size, there are important exceptionsto this pattern.

2. Surface-Volume Ratio- With the growth of the cell size, its volume increases more than
its surface area. All the materials of the cell required for its maintenance and growth are
drawn through its surface. A stage will reach when the surface area is insufficient to supply
the large volume of the cell. It is thought that there is a critical point at which the cell
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division starts and the division of the cell greatly increases the surface without increasing
the volume. This theory fails in case of starved cells, whichmay divide without doubling
their size and form smaller daughter cells.

3. Nucleolus- Damage to nucleolus at a certain critical time (telophase or mid prophase)

stops cell division.

4. Cyclic Nucleotides- Concentration of cAMP and cGMP vary regularly during the cell
division. Concentration of cAMP is high during G 1 phase, but it falls as the cell entersthe S
phase and mitosis. However the concentration of cGMP often varies in the reverse pattern.
Thus, addition or removal of any of these nucleotides can start or stop entry of many cells
into S phase and the subsequent M phase. The concentration of these cyclic nucleotides
remains constant throughout the cell cycle in many cells.

Also, plant cells do not have cyclic nucleotides. On the basis of these facts, cyclic AMP and GMP
are no longer thought to regulate the cell cycle.

5. Phosphorylation- During cell cycle the phosphate groups are added to the histone groups
particularly to H1 as the cell enters S phase, increases during M phase, and are removed on
the completion of mitosis before G1 starts. Phosphate groups are also added and removed
to non-histone proteins during cell cycle. Thus, it is believed that the changes in the
histones and non-histones may have a role in the control of cell cycle because these
proteins have been found to regulate the activity of genes in RNA transcription during
interphase.

6. Cyclin: The concentration of the protein called cyclin appears to control mitosis as it builds
up during interphase and is degraded during mitosis.

Mitosis:-

A German biologist Eduard Strasburger described mitosis for the first time in 1875.Same was
described later in 1879 by Walther Flemming who also termed it "mitosis" in 1882.

It is the most common method of cell division in eukaryotes that takes place in somatic cells of
the body and hence it is also known as somatic division. However in gonads it occurs in
undifferentiated germ cells. In plants it takes place in the cells of meristematic tissues. The
duration of mitosis on an average is from 30 minutes to 3 hours. Mitosis is defined as the division
of a parent cell into two identical daughter cells eachwith a nucleus having the same amount of
DNA, the same number and kind of chromosomes and the same hereditary instructions as the
parent cell. Therefore, it is also known as the equational division. There are two main events
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involved in mitosis: Karyokinesis or divisionof the nucleus and cytokinesis or division of

cytoplasm.

Karyokinesis

In eukaryotes, karyokinesis is a complex process due to the presence of many

chromosomes. It is a continuous process which may be divided into four stages: prophase,
metaphase, anaphase and telophase.

Stages of mitosis in animal cells

1. Prophase- In an interphase cell the chromosomes are greatly extended and spread
throughout the space in the nuclear compartment. Approximately 4 meters of DNA is
organized into 46 duplicated chromosomes is present in the nucleus of a human G 2 cell.
The prophase is long and complex that lasts for about 50 minutes. It may be divided into
3 sub stages: early prophase, middle prophase and late prophase.

A) Early prophase- During the early prophase of mitosis the following events take place:

(i) The shape ofcell becomes almost rounded and the cytoplasm becomes viscous.

(ii) The centrioles lie close to the nucleus and around them assembles the short radiating
microtubules by polymerization of the tubulin dimers. Both pairs of centrioles also called
diplosomes, start moving to the opposite ends of the cell.The microtubules surrounding
each pair of centrioles appear like a star body, and are called the aster. The microtubules
which are also termed as astral rays, are not in contact with the centrioles, but are
separated from them by an amorphous zone of cytoplasm known as pericentriolar cloud.
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The microtubules stretching between the diplosomes moving apart increase in number
and length by incorporating more tubulin dimers. Thus, asters shift the duplicated
centrioles to the opposite ends of the cell from where the centriole pair will pass into
separate daughter cells when cytokinesis occurs. Though thecentrioles have no role in the
formation of the spindle but they may be concerned with orienting the spindle.

(iii) Long microtubules assemble on one side of the nucleus to form mitotic spindle.
Microtubules are arranged in bundles called spindle fibers and at each pole of the
spindle lies the mother-daughter centriole pair.

(iv) The chromosomes that appear like threads in the nucleus gradually change into short,
thick rods by loss of water and progressive coiling and become visible. Due to the
duplication of DNA and chromosomal proteins during the interphase, each chromosome
appears longitudinally double, consisting of two identical sister chromatids which are held
together at the narrow region called primary constriction or centromere. Each
chromatid has a disc like structure at centromere, where the spindle microtubules join
it. This disc is called as kinetochore.

B) Middle prophase- It includes the following events:

(i) The chromosomes further get shorter, thicker and their chromatids become uncoiled and
finally they assume their characteristics sizes and become distinguishable individually.

(ii) Nucleoli progressively become smaller and finally disappear. Nuclear envelope begins
to breakdown into small vesicles which disperse into the cytoplasm. The lamina
dissociates into its protein subunits.

C) Late Prophase- This phase involves the following events:

(i) The nuclear envelope breaks completely thus, releasing the chromosomes and other
nuclear contents into the cytoplasm.

(ii) The spindle gains their proper shape and size.

(iii) The growing spindles push the centriole pairs to the opposite ends of the cell.

2. Metaphase- The metaphase being short and simple lasts for 2 to 10 minutes and it
involves the following events:

A. The spindle occupies the region of the nucleus

B. The chromosomes move to the equatorial plane of the spindle.

C. Some spindle microtubules extend to and join the chromosomes. These are called
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chromosomal or kinetochore microtubules.

D. The chromosomes get aligned at the middle of the spindle in the form of a plate called
equatorial or metaphase plate. This plate is formed by thekinetochores, the arms of the
chromatids trailing away on the sides. It is at the right angles of the long axis of the spindle.
During metaphase the chromosomes have fully aligned into a plate and await the
separation of their chromatids.

3. Anaphase- Anaphase lasts only 2 to 3 minutes and it comprises the following events:

A. The sister chromatids of each chromosome slightly separate at the primary

constriction so that their kinetochores stretch towards the opposite poles of thespindle.
In all the chromosomes separation of chromatids occurs almost simultaneously. The
chromatids are now referred to as chromosomes because they are no longer held to
their duplicates.

B. After a short time, the chromatids separate completely from their formermates, and start
moving to opposite poles of the spindle. As each chromosome is being pulled by its
attached microtubules, its kinetochore leads and arms trail behind. As a result the
chromosomes are pulled into V, J and I shapes, depending upon the position of the
kinetochore. (Metacentric, sub metacentric or telocentric respectively)

C. As the chromosomes move toward their respective poles, the two poles move farther apart
by elongation of spindle.

The anaphase ends when all the chromatids reach the opposite poles. Each pole of the spindle
receive one chromatid from every metaphase chromosome, the two groups of chromatids
have exactly the same hereditary information.

4. Telophase- The telophase is long and complex and lasts for an hour or so. In this phase
nucleus is reconstructed from each group of chromosomes. It involves the following
events:

A. The chromosomes at each pole unfold, and become long and slender. Finally, they
become indistinguishable as were in an interphase cell.

B. Nuclear envelope is reconstructed around each group of chromosomes gradually. First,

the membrane vesicles associate with the individual unfolding chromosomes, partially
enclosing each chromosome. Then they fuse to form an envelope surrounding the entire
set of chromosomes at each pole. The lamina proteins re-associate simultaneously with
the reconstruction of nuclear envelope and form a complete lamina within the nuclear
envelope
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C. Nucleolar material, composed of partially processed ribosomal subunits and processing

enzymes, dispersed into the cytoplasm in the prophase return to the nucleolar organizer
site and forms a small nucleolus. Processing of this preexisting material then continues.
Transcription of new rRNA also begins at this time; it gradually speeds up until it attains
the high level of characteristic of interphase cell. Along with this, the nucleolus grows and
attains its normal size. The nucleolus reformed at telophase, thus contains both old and
new rRNA and ribosomal proteins.

With the transformation of chromosomes into chromatin and reconstruction of nucleoli,

transcription of all the three RNA types gradually becomes normal.

The spindle begins to disappear and the asters become small by depolymerization of
microtubules and the centrioles take up their characteristic interphase position close to
the one side of the nucleus. Short spindle microtubules persist for sometime at the spindle
equator to mark the region where the cytoplasm will later divide.

Cytokinesis:-

Cytokinesis is the division of cytoplasm. It encloses the daughter nuclei formed by the
karyokinesis in separate cells, thus completing the process of cell division. Cytokinesis is
signaled at the metaphase by cytoplasmic movements that bring about equal distribution
of mitochondria and other cell organelles in the two halves of the cell. Division occurs
differently in animal cells and the plant cells.
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Significance of Mitosis:-

 Maintenance of Size- Mitosis helps maintaining the size of the cell. A cell, when full grown,
divides by mitosis instead of growing further.

 Growth- A fertilized egg develops into an embryo and finally into an adult by repeated
mitotic cell division.

 Maintenance of Chromosome Number- Mitosis keeps the number of chromosomes

equal in all the cells of an individual. Thus mitosis provides a complete set of genetic
information to each cell, since DNA is duplicated in S phase prior to mitosis.

 Repair- Mitosis provides new cells to replace the old worn out and dying cells.

 Healing and Regeneration- Mitosis produces new cells for the healing of woundsand
regeneration.

 Reproduction- Mitosis brings about multiplication in the acellular organisms. In

multicellular organisms also, it plays an important role in reproduction, asexual as
well as sexual.

 Evidence of Basic Relationship of Organisms- Mitosis, being essentially similar in many

kinds of organisms, supports the basic relationship of all living things.

Meiosis:-

In 1887, August Weismann predicted on theoretical grounds that the number ofchromosomes
must be reduced by one-half during gamete formation. Edouard Van Benedendemonstrated
reduction division in1887. J.B. Farmer and Moore introduced the term "meiosis" in
1905.Mitosis occurs in all kinds of eukaryotic cells, while meiosis is confined to certain cells and
takes place at a particular time. Only the cells of sexually reproducing organisms undergo
meiosis, and only special cells in the multicellular organisms switch over from mitosis to meiosis
 108

at the specific time in the life cycle. Meiosis produces gametes or gametic nuclei in animals, some
lower plants, and various protists and fungus groups. Meiosis forms spore in higher plants. The
spores give rise to gamete producing structure called gametophytes, which produces gametes
by mitosis.

Meiosis consists of two divisions that take place in rapid succession, with the chromosomes
replicating only once. Thus, a parent cell produces four daughter cells, each having half the
number of chromosomes and half of the nuclear DNA amount present in the parent cell. Meiosis
is therefore also known as reduction division. The two divisions of meiosis are known as the
first and the second meiotic divisions or meiosis-I and meiosis-II.

Stages of meiosis in animal cells

Divisions of Meiosis:-

First meiotic division or Meiosis-I :-

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During the first meiotic division, the two homologous chromosomes of each pair separate from
each other and go to separate daughter cells. This reduces the number of chromosomes
from diploid to haploid condition. Meiosis-I is therefore known as heterotypicdivision.
The four phase of this division are called Prophase-1, metaphase-1, anaphase-1 and
telophase-1.

1. Prophase--. The meiotic prophase-1 is more complex than the mitotic prophasebecause
of the process of recombination that occurs in it. It also lasts much longer than the mitotic
prophase in the same organism. It may extend over weeks, months or

even years. Although it is more or less a continuous process, it is divided into 5 sub- stages:
leptotene, zygotene, pachytene, diplotene and diakinesis.

(a) Leptotene- Leptotene begins when chromosomes appear as thin threads by condensation.
The chromosomes become thicker as condensation proceeds. They lie jumbled up so that
it is not possible to trace individual chromosomes. Each chromosome is double, consisting
of two chromatids due to DNA replication during premeiotic interphase. However, the
chromatids are closely adhered together and are not distinguishable.

(b) Zygotene- The homologous chromosomes come to lie side by side in pairs. The pairing of
homologous chromosomes is called Synapsis or conjugation. A pair of homologous
chromosome lying together is termed as a bivalent. Pairing is so through that the
corresponding ends and all the corresponding genes of the two homologous chromosomes
lie exactly opposite to each other. The centrosome of the chromosomes also lies adjacent
to one another. The chromatids are still not visible. A regular space of about 0.15 to 0.2 µm
wide exists between the synapsed homologous chromosomes, bearing a highly specialized
fibrillar organelle, the synaptonemal complex. The synaptonemalcomplex consists of
three parallel and equally spaced longitudinal filaments flanked by chromatin and
interconnected by short transverse filaments. The complex contains DNA and some
specific proteinaceous material. It was discovered by Montrose J. Moses in 1955 in
crayfish.

Synaptonemal complex
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(c) Pachytene- The synapsed chromosomes continue to become short and thick. The
chromatids of each synapsed chromosome slightly separate and become visible. A
chromosome with two visible chromatids is known as dyad. A group of four homologous
chromatids (two dyads) is called a tetrad. The number of tetrads equals the haploid
number of chromosomes. The two chromatids of the same chromosomes are called are
called sister chromatids and those of the two homologous chromosomes are called non-
sister chromatids. The leptotene and the zygotene stages last for a few hours, the
pachytene may take weeks, months or even years. It is prolonged because recombination
or crossing over occurs in it.

Recombination involves mutual exchange of the corresponding segments of non-sister

chromatids of homologous chromosomes. It occurs by breakage and reunion of non sister
chromatid segments. Certain structures mediate the meiotic recombination by marking
the sites of crossing over. These are known as recombination nodules (RNs). They are
multicomponent proteinaceous ellipsoids found in association with the synaptonemal
complex during prophase-I of meiosis (Carpenter, 1975b). The synaptonemal complex,
aprotein structure, helps in recombination by keeping the homologous chromosomes in
paired state for the required period and also by containing and aligning the enzymes
needed for breakage and union.

(d) Diplotene- At this stage the homologous chromosomes separate at many places. This is
called disjunction. It occurs because the synaptic forces and the synaptonemal complex
disappear. The chromatids become more distinct and tetrads seem very clear. The
homologous chromosomes do not separate at certain points. These points are called
chiasmata. The chiasmata mark thesites where the exchange of chromatids occurred
during pachytene. The number of chiasmata is related to the length of the chromosomes.
Longer chromosomes have more chiasmata than the shorter ones. In case of single
chiasmata, the bivalent looks like a cross; in case of two chiasmata, it looks like a ring; and
in case of many it shows series of loops.

(e) Diakinesis- In this stage the chromosomes condense again into short, thick rods. The
chiasmata disappear by sliding towards the tips of chromosomes due to tight
condensation. This process is called terminalization. The centrioles already duplicated in
premeiotic interphase, move apart in pairs to the oppositeends of the cell. Asters form
around each centriole pair. Spindle develops between the centriole pairs. The nucleolus
disintegrates. The nuclear envelope breaks down into vesicles. The tetrads are released
into the cytoplasm.

2. Metaphase- The spindle shifts to the position that is earlier occupied by the nucleus. The
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tetrads scattered in the cytoplasm move to the equator of the spindle. Here, they align in
two parallel metaphase plates, one formed by chromosomes and other by their
homologous. The attachment of the tetrads to the spindle microtubules in metaphase-I is
different from that of mitotic metaphase chromosomes. Each homologous chromosome
has two kinetochores, one for each of its two chromatids. Both the kinetochores of a
homologous chromosome connect to the same spindlepole. The two kinetochores of its
homologue join the opposite spindle pole.

3. Anaphase-I- From each tetrad, two chromatids of a chromosome move as a unit (dyad) to
one pole of the spindle, and the other two chromatids of its homologue migrate to the
opposite pole. Thus, the two homologous chromosomes of each pair are separated in the
anaphase-I of meiosis. The process is also called as disjunction. As a result half of the
chromosomes, which appear in early prophase, go to each pole. Thus, it is during
anaphase-I that the real reduction in the chromosome number occurs. Each chromosome
at the pole is still double and consists of two chromatids. Thus, the group of chromosomes
at each pole though has only one member of each homologous pair still contains twice the
haploid amount of DNA.

4. Telophase-During telophase-I, the chromosome at each pole of the spindle partly unfold
and elongate, and form a nucleus with nucleolus and nuclear envelope. The spindle and
asters disappear.

The cytoplasm divides at its middle by constriction in an animal cell and by cell plate formation
in a plant cell. This produces, two daughter cells, each with one nucleus. The nucleus of
each daughter cell has received only one chromosome from each homologous pair. Thus,
it has half the number of chromosome, but double the amount of nuclear DNA as each
chromosome is double.

First meiotic division or Meiosis-II

The meiosis-II is similar to mitosis as in this division, the two chromatids of each chromosome
separate from each other and go to separate daughter cells. With the result, the number of
chromosomes remains the same as produced by meiosis-I. Meiosis-II is, therefore, known
as homotypic division. The four stages of this division are called prophase-II, metaphase-
II, anaphase-II and telophase-II.

1. Prophase-I- When there is no interkinesis, the telophase-I spindle is replaced by two new
spindles; and the centrioles and asters, if present, duplicate and one copy of each comes
to lie at each pole of the new spindles. The telophase-I chromosomes move from the poles
of the old spindle to the equators of the new spindles. If decondensation has occurred
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during telophase-I, the chromosome recondense to short rod lets as they migrate to the
metaphase-II spindles.

If interkinesis is present, centrioles move apart and asters are formed around them. A spindle is
formed between the centrioles. Chromosomes each consisting of two chromatids, appear
in the nucleus. They are set free in the cytoplasm by breakdown of the nuclear envelope.
Nucleus disappears.

2. Metaphase-II- The chromosomes get arranged at the equator of the spindle as a

metaphase plate. The chromatids of each chromosome are joined at their kinetochores by
chromosomal microtubules extending from the opposite poles of the spindle as in mitosis.

3. Anaphase-II- The two chromatids of each chromosome separate and move to the opposite
poles of the spindles. Here they are called chromosomes. Each pole has haploid number
of chromosomes and haploid amount of DNA. This amount is one-fourth of the DNA
present in the original cell which entered meiosis.

4. Telophase-I-: The chromosome at each pole decondenses, and nuclear envelopedevelops

around them. This produces two nuclei. Nucleolus is formed in each nucleus. Spindle and
asters disappear. In cases that lack interkinesis, four nuclei are formed in telophase-II.

Cytokinesis:-

Cytoplasm divides at its middle by constriction in an animal cell and by cell plate formation in a
plant cell. This produces two daughter cells. The later have half the number of chromosomes,
and half the amount of nuclear DNA, i.e., in Reduction division is complete when this point is
reached. The cells formed by meiosis-II in animals are mature gametes. They do not divide
further. A gamete must fuse with another suitable gamete before a new individual can develop.
The cells formed by meiosis-II in plants are the spores. The sporescan develop into new
individuals without fusing in pairs. In fact the main difference betweena spore and a gamete is
the ability of the spore to develop directly into a new individual.

Comparison between Mitosis and Meiosis

Mitosis and meiosis can be differentiated through following points:-

Mitosis Meiosis
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It occurs in all kinds of cells and may It occurs only in special cells (gametemother
continue throughout life cells or spore mother cells) and at specific
times
It involves a single division, resulting in It involves two successive divisions, resulting
two daughter cells only in four daughter cells
A cell can repeat mitosis almost in Meiosis takes place only once in a cell
definitely
All mitotic divisions are alike Two meiotic divisions are dissimilar, first is
reductional and second equational
Each mitotic division is preceded by an The second meiotic division is generally not
interphase preceded by an interphase
Chromosomes replicate before each Chromosomes do not replicate before each
mitotic division mitotic division
Prophase is relatively short and simple Prophase-1 is very long and elaborate,
comprising 5 sub phases
Prophase chromosomes appear double Prophase-1 chromosomes do not look
from the very start. double in the beginning
There is no pairing of homologous Homologous chromosomes pair and often
chromosomes,hence no chance of crossing undergo crossing over in prophase-1.
over.
No chiasmata are formed Chiasmata form temporarily where crossing
over occurs
Chromatids are genetically similar to Chromatids may differ genetically from the
chromosomes they arise from chromosomes they arise from due to
crossing over.
No synaptonemal complexforms between Synaptonemal complex forms between
chromosomes synapsed homologous chromosomes
Chromosomes do not unfold, and no Chromosomes unfold and, transcription and
transcription and protein synthesis occur protein synthesis may occur in diplotene of
in prophase prophase-I.
All chromosomes form a single plate in Chromosomes form two parallel plates in
metaphase. metaphase-I and one plate in metaphase-II.
The two kinetochores of a chromosome The kinetochores of a chromosome connect
connect to both the poles of the spindle. to the same spindle pole in metaphase-I and
to both the poles in metaphase-II.
Anaphase involves separation of Anaphase-I involves separation of
chromatids of each chromosome. homologous chromosomes. The chromatids
move apart in anaphase-II.
Telophase occurs in all cases Telophase-I is eliminated in some cases.
Daughter cells have diploid number of Daughter cells have haploid number of
chromosomes like the parent cell. chromosomes unlike the parent cell.
Daughter cells have 2n amount of DNA Daughter cells have 1n amount of DNA unlike
unlike 4n amount in parent cells the 4n amount in the parent cell
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Daughter cells divide again after Daughter cells, if gametes, do not divide
interphase. further.
Mitosis brings about growth,repair and Meiosis forms gametes or spores, helps
healing maintain the number of chromosomes
constant from generation to generation, and
introduces variation
Mitosis is much shorter than meiosis in Meiosis is much longer than mitosis in the
the same animal same animal.
Cytokinesis usually follows karyokinesis Cytokinesis often doesn't occur after meiosis-
I, but always occur after meiosis-II, forming
four cells simultaneously.
Mitosis may occur in haploid or diploid Meiosis always occurs in diploid cells
cells
Chromosomes do not show Chromosomes may showchromomeres.
chromomeres.

Cell signalling

One characteristic common to all organisms is the dynamic ability to coordinate constantly their
activities with environmental changes. The function of communicating with the environment is
achieved through a number of pathways that receive and process signals originating from the
external environment, from other cells within the organism and also from different regions
within the cell.

In addition to adapting the function of an organism to environmental changes in a signal-

directed way, other essential features of multicellular organisms require the coordinated control
of cellular functions as well.

The formation and maintenance of the specialized tissues of multicellular organisms depend on
the coordinated regulation of cell number, cell morphology, cell location and expression of
differentiated functions. Such coordination results from a complex network of communication
between cells in which signals produced affect target cells where they are transduced into
intracellular biochemical reactions that dictate the physiological function of the target cell. The
basis for the coordination of the physiological functions within a multicellular organism is
intercellular signaling (or intercellular communication), which allows a single cell to influence
the behavior of other cells in a specific manner. As compared to single-cell organisms, where all
cells behave similarly within a broad frame, multicellular organisms contain specialized cells
forming distinct tissues and organs with specific functions. Therefore, higher organisms have to
coordinate a large number of physiological activities such as:

– Intermediary metabolism.
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– Response to external signals.

– Cell growth.
– Cell division activity.
– Differentiation and development: coordination of expression programmes.
– Cell motility.
– Cell morphology

Signals generated during intercellular communication must be received and processed in the
target cells to trigger the many intracellular biochemical reactions that underlie the various
physiological functions of an organism. Typically, many steps are involved in the processing of
the signal within the cell, which is broadly described as intracellular signaling. Signal
transduction within the target cell must be coordinated, fine-tuned and channeled within a
network of intracellular signaling paths that finally trigger distinct biochemical reactions and
thus determine the specific functions of a cell. Importantly, both intercellular and intracellular
signaling are subjected to regulatory mechanism that allow the coordination of cellular functions
in a developmental- and tissue-specific manner

Tools for Intercellular signalling

Currently know of various forms of communication between cells:

– Extracellular messengers. Cells send out signals in the form of specific messenger molecules
that the target cell transmits into a biochemical reaction. Signaling cells can simultaneously
influence many cells by messenger molecules so as to enable a temporally coordinated reaction
in an organism.
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– Gap junctions. Communication between bordering cells is possible via direct contact in the
form of “gap junctions”. Gap junctions are channels that connect two neighboring cells to allow
a direct exchange of metabolites and signaling molecules between the cells.

– Cell–cell interaction via cell surface proteins. Another form of direct communication between
cells occurs with the help of surface proteins. In this process a cell surface protein of one cell
binds a specific complementary protein on another cell. As a consequence of the complex
formation, an intracellular signal chain is activated which initiates specific biochemical reactions
in the participating cells. Communication is then only possible upon direct contact between the
target cell with the surface protein of the partner cell.

– Electrical signaling. A further intercellular communication mechanism relies on electrical

processes

Principal mechanisms of intercellular communication. (a) Communication via intercellular

messengers. (b) Communication via gap junctions. Gap junctions are direct connections between
cells. They are coated by proteins (drawn as circles) that can have a regulatory influence on the
transport. (c) Communication via surface proteins

Formation of a Signal in the Signal-producing Cell as a Result of an External Trigger

Most extracellular messengers are produced in response to external triggers and are released
by exocytosis. Physical stimuli like electrical signals, changes in ion concentration or, most
frequently, other extracellular signaling molecules serve as a trigger to increase the amount of
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the messenger available for extracellular communication. The mechanisms by which the
external trigger signals increase the amount of extracellular messenger are diverse, and include
stimulation of the biosynthesis of the messenger, increased production of the mature messenger
from precursors and release of the messenger from a store form. The latter mechanism is
extensively used in the release of hormones of the neural system (neurotransmitters) in
response to electrical signals, e.g. at synapses.

Transport of the Signal to the Target Cell

The extracellular signal produced may be distributed via the circulation or it may reach the
target cell simply by diffusion. In long-range signaling via the circulation, the extracellular
messenger is often bound to specific carrier proteins or incorporated into larger protein
complexes. This may serve to prevent degradation in the extracellular medium or to provide for
docking to specific cells only. Furthermore, processing or metabolization of a messenger during
transport may convert it from an inactive form to an active form.

Registration of the Signal in the Target Cell

A target cell that receives a signal within the framework of intercellular communication
transmits the signal in intracellular pathways that trigger distinct biochemical activities in a cell-
type-specific manner and determine the response of the target cell. Specialized proteins, termed
receptors, are utilized for the reception of signals in the target cell. Only those cells that carry
the appropriate receptor will be activated for further transduction of the signal into the interior
of the cell. The reception of the signals by the receptor is equivalent to the binding of messenger
substance on the receptor or the transmission of physical stimuli into a structural change in the
receptor.

Endocrine, Paracrine and Autocrine Signaling

Endocrine Signaling

In endocrine signaling, the hormone messenger is synthesized in specific signaling, or endocrine,

cells and exported via exocytosis into the extracellular medium (e.g. blood or lymphatic fluid in
animals). The hormone is then distributed throughout the entire body via the circulatory system
so that remote regions of an organism can be reached. Only those cells or tissues elicit a
hormonal response that contain the appropriate receptor for the hormone.

Paracrine Signaling

Paracrine signal transduction occurs over the medium range. The hormone reaches the target
cells from the hormone-producing cell by passive diffusion. The producing cell must be found in
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the vicinity of the receiving cells for this type of communication. The signaling is rather local and
the participating signaling molecules are sometimes termed tissue hormones or local mediators.
A special case of paracrine signal transduction is synaptic neurotransmission in which a nerve
cell communicates with either another nerve cell or with a muscle cell.

Autocrine Signaling

In autocrine signaling, cells of the same type communicate with one another. The hormone
produced by the signaling cell affects a cell of the same type by binding to receptors on these
cells, initiating an intracellular signal cascade. If an autocrine hormone is secreted
simultaneously by many cells then a strong response occurs in the cells. Autocrine mechanisms
are of particular importance in the immune response.

Cell adhesion

The construction of signaling proteins from distinct protein domains and the assembly of
signaling proteins into larger signaling complexes results in multifunctionality, variability and
interconnection of cellular signaling systems. It has been proposed that cells contain a general
signaling network that may be operationally divided into large signaling modules or types of
signaling paths. The signaling modules contain characteristic core components and are defined
by functional input–output characteristics. Examples of such modules are the epidermal growth
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factor (EGF) receptor–Grb2–mSos– Ras module, the MAPK module and the G-protein-coupled
receptor (GPCR)–Gbg–Ga–adenylyl cyclase–cAMP protein kinase A (PKA) module (Fig. 1.14a).
The molecular identity of the components of the signaling modules and their interacting
partners may be cell type-specific, but the overall function of these components and the logic of
the circuitry is preserved from cell type to cell type. The central signaling system is connected
to cellular mechanisms such as the transcriptional, translational, motility and secretory
machinery that are responsible for phenotypic functions. The central network that connects the
various machine networks also receives and processes signals from extracellular entities such
as hormones or neurotransmitters and ions.

Signal transduction through signaling networks depends on the types of interconnection of its
components and the properties of the components itself. The large number of possible
connections and the multiplicity of possible input and output signals at each component makes
the description of signaling networks extremely complex. Nevertheless, one would like to
understand how precise signals leading to defined biochemical reactions can be produced in the
cell, how signals can be organized in time and in space, how irreversible switches are generated,
and how signals are dissipated and downregulated.

Gap Junction
 120

The interconnections in signaling networks may be operationally divided into two classes:
junctions, which are signal integrators, and nodes, which split the signal. This classification is,
however, by no means absolute, because we know of quite a number of signaling proteins that
can receive and distribute multiple signals

The node function of the Rho-GTPase CDC42. This small regulatory GTPase (Section 9.9) can
receive signals from several TM receptors and can deliver signals to different protein kinases
and to transcription factors.

A well studied example of a system that contains both junctions and a node is the cAMP/PKA
system. Here, the various subtypes of adenylyl cyclase function as signal integrators or signaling
junctions that receive signals from various TM receptors or ion channels. The adenylyl cyclases
transduce the signal to PKA, of which only few isoforms exist. The PKA functions as a node
through which the signal is distributed to various downstream partners. Signal distribution by
PKA is achieved by the differential use of isoforms of the adaptor proteins [A-kinase anchoring
proteins (AKAPs)] that specify the nature and location of the substrate of the protein kinase.

Networks also contain nodes where signals may be split and routed through several different
pathways to regulate distinct cellular functions. Like junctions, nodes may also be upstream or
downstream in the network. One of the best upstream examples of a node is the RTKs, which can
route growth factor signals through many different pathways. Although such routing can result
in regulation of multiple independent cellular functions (e.g. growth factors such as PDGF can
regulate vascular smooth muscle cell migration and proliferation), signal routing through
multiple pathways can produce combinatorial signal specificity at the level of gene expression.
121
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Unit V

CENTRAL DOGMA

In 1958 F.Crick proposed that the concept of central dogma, which states that when a
particular gene is expressed (control a function or a reactions) its information is copied into
another nucleic acid (mRNA) which in turn directs the synthesis of specific proteins. So the
central dogma was proposed as unidirectional flow of molecular information from DNA to mRNA
and finally to polypeptide. Later a reverse of central dogma was also found in retroviruses. H.
Temin and D. Baltimore (1970) reported that retro viruses operate a central dogma in reverse
manner (inverse flow of information) or teminism inside host cells. Thisdiscovery was

important in understanding cancer and hence, these two scientists wereawarded Nobel Prize.

Linear flow of Central Dogma and Reverse of Central Dogma

Genetic RNA of these viruses first synthesizes DNA through reverse transcription. This process
is catalyzed by the enzyme reverse transcriptase. DNA then transfers information tomessenger
RNA which takes part in translation of the coded information to from polypeptide

Stahl experiment

The Meselson- Stahl Experiment- The result of the first critical test of Watson and Crick’s
proposal that DNA replicates semi conservatively were published in 1958 by M.S. Meselson and
F.W. Stahl. Their experiment was as follows:

 They grew Escherichia coli for many generations in a medium having heavy isotopes

of nitrogen, N15isotope. Till the bacterial DNA becomes completely labelled with heavy

 The labeled bacteria were then shifted to fresh medium having normal or N14.

 After each cell division DNA was separated from a sample of the cells and analyzed
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on a CsCl (cesium chloride) gradient using the technique of equilibrium density gradient
centrifugation, which separates molecules according to differences inbuoyant density.

 Meselson and Stahl found that DNA of the first generation was hybrid or intermediatebetween
N15 and N14.

 The second generation of bacteria contained two types of DNA, 50% light and 50%hybrid.

 There were exactly the results to be expected if DNA replication is semi conservative

Diagram of Semi-conservative Replication.

[After M. Meselson and F. W. Stahl. Proc. Natl. Acad. Sci. U.S.A. 44(1958):671.]

Mechanism of DNA Replication:-

DNA replication is the process of copying a DNA molecule and involves following four
major steps-

1. Initiation of DNA replication

2. Unwinding of helix

3. Formation of primer strand

4. Elongation of new strand.

5. Termination

1. Initiation of DNA replication- Replication is regulated by the rate of initiation.

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Replication of DNA in E. coli always begins at a definite site called origin of replication. The E.
coli, origin of replication lies within the genetic locus ‘ori’ and is bond to the cell membrane. ‘Ori’
contains four 9bp binding sites for the initiator protein (DnaA-ATP). The helicase DnaB (or
mobilepromoter) binds and extends the single-stranded region for copying.

2. Unwinding of helix- Unwinding of DNA molecule into two strands results in the
formationof Y shaped structure called replication fork. Due to unwinding positive super coiling
has to be relieved by the enzyme topoisomerase or DNA Gyrase.

3. Formation of Primer strand- As the newly formed replication fork displaces the
parental lagging strand, a mobile complex called a primosome, which includes the DnaB,
Helicase and DNA primase help in the synthesizes of RNA primers. Both leading and lagging
strand primers are elongated by DNA polymerase III. Need of primer is there to facilitate the
action of DNA polymerase III as this enzyme cannot initiate the process but can add activated
deoxyribonucleotides to the 3’ OH end of primer.

4. Elongation of new strand – after the formation of primer strand, DNA replication
occurs in 5’ 3’direction and complementary deoxyribonucleotides are added only to the free
3’OH end of the primer. A dimer of DNA polymerase III elongates both leading (3’ 5’) and lagging
strands. The leading strand shows continuous replication while the lagging strand shows
discontinuous replication. These short pieces of DNA replicated against lagging strand are
known as Okazaki fragments. Okazaki fragments are 1000-2000 nucleotides long in
prokaryotes. A separate RNA primer is used for the synthesis of each Okazaki fragments which,
after replacing the RNA primers from deoxyribonucleotides, are later joined together with the
help of DNA ligase or DNA synthetase forming a continuous lagging strand. Hence DNA
replication is semi- discontinuous as the leading strand is synthesized continuously and the
lagging strand is formed discontinuously in short pieces that join later.
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5. Termination

The primers are removed and the gaps are filled with DNA Polymerase I and sealed by ligase.

Termination occurs when a termination site sequence in the DNA is reached, and a protein binds
to this sequence to physically stop DNA replication, the DNA replication terminus site-binding
protein, Ter protein, an inhibitor of DnaB helicase which works along with a Tus factor (forming
a complex).

Important features of Prokaryotic replication

1. Bacteria have a single loop of DNA that must replicate before the cell divides.

2. Replication proceeds in one direction from 5’- 3’.

3. Replication may be bidirectional or directional.

4. One cycle of DNA replication gets completed in 40 minutes.

5. Prokaryotes are able to replicate their DNA at a rate of about 106 base pairs/min

Important features of Eukaryotic replication

i. Replication starts at many points of origin and spreads with many replication bubbles.
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These bubbles are the places where the DNA strands are separating and replication is
occurring.

ii. Replication forks are the V shaped ends of the replication bubbles.

iii. Eukaryotes replicate their DNA at slower 500-5000 base pairs per minutes.

iv. These cells can complete DNA replication in one hour

Okazaki fragments

Whereas the DNA polymerase III on the leading strand can simply follow the replication fork,
because DNA polymerase III must move in the 5' to 3' direction, on the lagging strand the enzyme
must move away from the fork. The lagging strand replicates in small segments, called Okazaki
fragments. These fragments are stretches of 100 to 200 nucleotides in humans (1000 to 2000 in
bacteria) that are synthesized in the 5' to 3' direction away from the replication fork.

For synthesis of each Okazaki fragment a new primer is made before the segment is replicated.
After replication of over these primers are degraded. Now in the vacant spaces DNA is
synthesized by DNA polymerase I. These fragments are then stitched together by DNA ligase,
creating a continuous strand. The synthesis of lagging strand is called discontinuous

RNA: STRUCTURE, FUNCTION AND TYPES:

Ribonucleic acid (RNA) is a polymeric molecule essential in various biologicalroles in coding,

decoding, regulation and expression of genes. RNAand DNA are nucleic acids, and, along with
lipids, proteins and carbohydrates, constitute the four major macromolecules essential for all
known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is
more often found in nature as a single-strand folded onto itself, rather than a paired double-
strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the
nitrogenous bases of guanine, uracil, adenine, and cytosine denoted by the letters G, U, A, and C)
that directs synthesis of specific proteins.

Many viruses encode their genetic information using an RNA genome.

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Secondary Structure of RNA - Stem-loop and Hairpin.

There are more unusual bases in RNAthan in DNA. All normal RNAchains either start with adenine or
guanine: Threetypes of cellularRNA have beendistinguished:

1. Messenger RNA (mRNA) or template RNA

2. Ribosomal RNA (rRNA) and

3. Soluble RNA (sRNA) or transfer RNA (tRNA)

Ribosomal and transfer RNAcomprise about 98% of all RNA.All three forms ofRNA are made on a DNA
template. Transfer RNA and messenger RNA are synthesized on DNA templates of the
chromosomes, while ribosomal RNA isderived from nucleolar DNA. The three types of RNA are
synthesized during different stages in earlydevelopment. Most of the RNA synthesized during cleavageis
mRNA. Synthesis of tRNA occurs at the end or cleavage, and rRNA synthesisbegins during gastrulation.

Ribosomal RNA – rRNA

Ribosomal RNA, as the name suggests, is found in the ribosomes. It comprisesabout 80% of the total
RNA of the cell. The base sequence of rRNA is complementaryto that of the region of DNA where it is
synthesized. In eukaryotes ribosomes are formed on the nucleolus. Ribosomal RNA is formed from
only asmall section of the DNA molecule, and hence there is no definite base relationshipbetween rRNA
and DNA as a whole. Ribosomal RNA consists of a single strandtwisted upon itself in some regions. It has
helical regions connected by interveningsingle strand regions. The helical regions may show presence or
absence of positive interaction. In the helical region most of the base pairs are complementary, andare
joined by hydrogen bonds. In the unfolded single strand regions the baseshave no complements.
Ribosomal RNA contains the four major RNA bases with a slight degree of methylation, and shows
differences in the relative proportions of the bases between species. Its molecules appear to be single
polynucleotide strands which areunbranched and flexible. At low ionic strength rRNA behaves as a
random coil, but with increasing ionic strength the molecule shows helical regions produced by base
pairing between adenine and uracil and guanine and cytosine

Hence rRNAdoes not show purine-pyrimidine equality.The rRNAstrands unfold upon heatingand refold
upon cooling.Ribosomal RNA is stable for at leasttwo generations. The ribosome consists of proteins and
RNA. The 70S ribosomeof prokaryotes consists of a 30S subunit and a 50S subunit. The 30S subunit
contains 16S rRNA, while the 50S subunit contains 23S and 5S rRNA. The 80S eukaryote ribosome
consists of a 40S and a 60S subunit. In vertebrates the 40Ssubunit contains 18S rRNA, while the 60S
subunit contains 28- 29S, 5.8S and5S rRNA. In plants and invertebrates the 40S subunit contains
16- 18S RNA,while the 60S subunit contains 25S and 58 and 5.8S rRNA.
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There are three types of ribosomal RNA on the basis of sedimentation and molecular weight. Two of
these classes are high molecular weight RNAs, whilethe third is a low molecular weight RNA. The three
classes are:

high molecular weight rRNA with molecular weight of over a million,for example 21s-29s RNA,

high molecular weight rRNA with molecular weight below a million,for example 12-8-188 rRNA,

low molecular weight rRNA, for example 58 rRNA.

Stability of Messenger RNA - mRNA

The cell does not contain large quantities of mRNA. This is because mRNA, unlike other RNAs is
constantly undergoing breakdown. It is broken down to its constituent ribonucleotides by
ribonucleases.

Structure of Messenger RNA

mRNA Messenger RNA is always single stranded. It contains mostly the bases adenine, guanine,
cytosine and uracil. There are few unusual substituted bases. Although there is a certain amount of
random coiling in extracted mRNA, there isno base pairing. In fact base pairing in the mRNA strand
destroys its biologicalactivity. Since mRNA is transcribed on DNA (genes), its base sequence is
complementary to that of the segment of DNA on which it is transcribed. This hasbeen demonstrated by
hybridization experiments in which artificial RNA, DNA double strands are produced. Hydrization
takes place only if the DNA and RNAstrands are complementary.

Usually each gene transcribes its own mRNA. Therefore, there are approximately as many types of
mRNA molecules as there are genes. There maybe 1,000 to 10.000 different species of mRNA in a
cell. These mRNA types differ only in the sequence of their bases and in length. When one gene
(cistron)codes for a single mRNA strand the mRNA is said to be monocistronic. In manycases, however,
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several adjacent cistrons may transcribe an mRNA molecule,which is then said to be polycistronic or
polygenic.

The mRNA molecule has the following structural features:

1. Cap: At the 5' end of the mRNA molecule in most eukaryote cells andanimal virus molecules is found
a‘cap’. This is blocked methylated structure,m7Gpp Nmp Np or m7Gpp Nmp Nmp Np. where: N = any
of the fournucleotides and Nmp = 20 methyl ribose. The rate of protein synthesisdepends upon the
presence of the cap. Without the cap mRNA moleculesbind very poorly to the ribosomes.

2. Noncoding Region 1 (NC1): The cap is followed by a region of 10 to100 nucleotides. This region
is rich in A and U residues, and does nottranslate protein.

3. The Initiation Codon: It is AUG in both prokaryotes and eukaryotes.

4. The Coding Region: It consists of about 1,500 nucleotides on the averageand translates protein It is
made up of 73-93 nucleotides (Rich and RajBhandary, 1976). Each bacterial cell probably contains
about a hundredor more different types of tRNA. The function of tRNA is to carry aminoacids to mRNA
during protein synthesis. Each amino acid is carried by aspecific tRNA. Since 20 amino acids are coded
to form proteins, it followsthat there must be at least 20 types of tRNA.

It was formerly thought that only 20 tRNA molecular types exist, one for each amino acid. It has,
however, been shown that in several cases thereare at least two types of tRNA for each amino acid.
Thus there are manymore tRNA molecules than amino acid types. These are probably codedby one
gene.

Transfer RNA is synthesized in the nucleus on a DNA template. Only0.025% of DNA codes for tRNA.
Synthesis of tRNA occurs near the endof cleavage stages. Transfer RNAis an exception to other cellular
RNAs inthat a part of its ribonucleotide sequence (-CCA) is added after it comesoff the DNA template.
Like rRNA, tRNA is also formed from only a smallsection of the DNA molecule. Therefore, it does not
show any obvious base relationships to DNA. The tRNA molecule consists of a single strand looped
about it self. The 3' end always terminates in a -C-C-A (cytosine-cytosine-adenine) sequence. The 5'
end terminates in G (guanine) or C(cytosine). Many of the bases are bonded to each other, but there
are alsounpaired bases.

Transfer RNA - tRNA OR Soluble RNA

sRNA After rRNAthe second most common RNA in the cell is transfer RNA. Itis also called soluble
RNA because it is too small to be precipitated by ultracentrifugation at 100,000 g. It constitutes
about 10-20% of the total RNA of the cell. Transfer RNA is a relatively small RNA having a molecular
weight of about 25,000 to 30,000 and the sedimentation coefficient of mature eukaryote tRNA is
3.8S.
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Structure of Transfer RNA – tRNA

The nucleotide sequence (primary structure) of tRNA was first worked out byHolley et al (1965) for
yeast alanine tRNA. Since then the sequence of about 75 different tRNAs, ranging from bacteria to
mammals, has been established. The different tRNAs are all minor variants of the same basic type of
structure. Several models of the secondary structure of tRNA have been proposed, and of these the
cloverleaf model of Holley is the most widely accepted.

Transfer RNA (tRNA) is an essential component of the protein synthesis reaction. There are at least
twenty different kinds of tRNA in the cell1 and eachone serves as the carrier of a specific amino acid to
the site of translation. tRNA’sare L-shaped molecules.

The amino acid is attached to one end and the other end consists of threeanticodon nucleotides. The
anticodon pairs with a codon in messenger RNA(mRNA) ensuring that the correct amino acid is
incorporated into the growingpolypeptide chain. The L-shaped tRNA is formed from a small single-
stranded RNA molecule that folds into the proper conformation. Four different regions of double-
stranded RNA are formed during the folding process

The two ends of the molecule form the acceptor stem region where theamino acid is attached. The
anticodon is an exposed single-stranded region in aloop at the end of the anticodon arm. The two
other stem/loop structures arenamed afterthe modified nucleotidesthat are found in those parts ofthe
molecule. The D arm contains dihydrouridylate residues while the T C arm contains a
ribothymidylate residue (T), a pseudouridylate residue ( ) and a cytidylate (C)residue in that order.
All tRNA’s have a similar T C sequence. The variable armis variable, just as you would expect. In some
tRNA’s it is barely noticable while in others it is the largest arm. tRNA’s are usually drawn in the
‘cloverleaf’ form(below) to emphasize the base-pairs in the secondary structure
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Cover Leaf Model of tRNA

Specificity of Tranfer RNA - tRNA

Two important steps in translation during protein synthesis are the activation ofamino acids and the
transfer of amino acids to tRNAs. Each amino acid has aspecific activating enzyme tRNA aminoacyl
synthetase. Thus there are 20 different tRNA aminoacyl synthetases for the 20 common amino acids
found in proteins.

Some tRNA synthetases can activate more than one amino acid, i.e. they show only a limited
substrate specificity. Thus isoleucine tRNA synthetase can also activate L valine, and valine tRNA
synthetase can also react with threonine. The enzymes, however, recognize only a specific set of,
tRNAs as substratesL isolecine tRNA synthetase recognizes only tRNAileu and valine tRNA synthetase
recognizes only tRNAval. Thus specificity is involved at two stages, activation of the amino acid and
transfer of the amino acid to tRNA.Another group of enzymes,the tRNA aminoacyl transferases catalyse
the transfer of an amino acid from theamino acid - tRNA complex to specific acceptor molecules.

RNA Synthesis - Transcription

Transcription in Prokaryotes

Enzyme involved

 RNA is synthesized by a single RNA polymerase enzyme which contains multiple polypeptide
subunits.

 In E. coli, the RNA polymerase has subunits: two α, one β, one β’ and one ω and σ subunit
(α2ββ’ωσ). This complete enzyme is called as the holoenzyme.

 The σ subunit may dissociate from the other subunits to leave a form known as the core
enzyme.
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Initiation Phase

During initiation, RNA polymerase recognizes a specific site on the DNA, upstream from the gene that
will be transcribed, called a promoter site and then unwinds the DNA locally.

Promoters and Initiation

 The holoenzyme binds to a promoter region about 40–60 bp in size and then initiates
transcription a short distance downstream (i.e. 3 to the promoter).

 Within the promoter lie two 6 base pair sequences that are particularly important for
promoter function.

 They are highly conserved between species.

 Using the convention of calling the first nucleotide of a transcribed sequence as +1, these two
promoter elements lie at positions –10 and –35, that is about 10 and 35 bp, respectively,
upstream of where transcription will begin.

 The –10 sequence has the consensus Because this element was discovered by Pribnow, it is
also known as the Pribnow box. It is an important recognition site that interacts with the σ
factor of RNA polymerase.

 The –35 sequence has the consensus TTGACA and is important in DNA unwinding during
transcriptional initiation.

 RNA polymerase does not need a primer to begin transcription; having bound to the
promoter site, the RNA polymerase begins transcription directly.

Elongation Phase

 After transcription initiation, the σ factor is released from the transcriptional complex to leave
the core enzyme (α2 ββω) which continues elongation of the RNA transcript.

 The core enzyme contains the catalytic site for polymerization, probably within the β subunit.

 The first nucleotide in the RNA transcript is usually pppG or pppA.

 The RNA polymerase then synthesizes RNA in the 5’ →3’ direction, using the four
ribonucleoside 5-triphosphates (ATP, CTP, GTP, UTP) as precursors.

 The 3-OH at the end of the growing RNA chain attacks the α phosphate group of the incoming
ribonucleoside 5-triphosphate to form a 3’5′ phosphodiester bond.

 The complex of RNA polymerase, DNA template and new RNA transcript is called a ternary
complex (i.e. three components) and the region of unwound DNA that is undergoing
transcription is called the transcription bubble.
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 The RNA transcript forms a transient RNA–DNA hybrid helix with its template strand but then
peels away from the DNA as transcription proceeds.

 The DNA is unwound ahead of the transcription bubble and after the transcription complex
has passed, the DNA rewinds.

 Thus, during the elongation, the RNA polymerase uses the antisense (-) strand of DNA as
template and synthesizes a complementary RNA molecule.

 The RNA produced has the same sequence as the non-template strand, called the sense (+)
strand (or coding strand) except that the RNA contains U instead of T.

 At different locations on the bacterial chromosome, sometimes one strand is used as

template, sometimes the other, depending on which strand is the coding strand for the gene
in question.

 The correct strand to be used as template is identified for the RNA polymerase by the
presence of the promoter site.

Termination Phase

 Transcription continues until a termination sequence is reached.

 The most common termination signal is a GC-rich region that is a palindrome, followed by an
AT-rich sequence.

 The RNA made from the DNA palindrome is self- complementary and so base pairs internally
to form a hairpin structure rich in GC base pairs followed by four or more U residues.

 However, not all termination sites have this hairpin structure. Those that lack such a structure
require an additional protein, called rho, to help recognize the termination site and stop
transcription.

 Thus the RNA polymerase encounters a termination signal and ceases transcription, releasing
the RNA transcript and dissociating from the DNA.
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Transcription in Eukaryotes

Transcription occurs in eukaryotes in a way that is similar to prokaryotes with reference to the basic
steps involved.

However, some major differences between them include:

 Eukaryotic Initiation is more complex.

 Eukaryotic Termination does not involve stem-loop structures.

 Eukaryotic Transcription is carried out by three enzymes (RNA polymerases I, II and III).

 The regulation of eukaryotic transcription is more extensive than prokaryotes.

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Enzyme involved in Eukaryotic Transcription

Unlike prokaryotes where all RNA is synthesized by a single RNA polymerase, the nucleus of a
eukaryotic cell has three RNA polymerases responsible for transcribing different types of RNA.

 RNA polymerase I (RNA Pol I) is located in the nucleolus and transcribes the 28S, 18S, and
5.8S rRNA genes.

 RNA polymerase II (RNA Pol II) is located in the nucleoplasm and transcribes protein-
coding genes, to yield pre-mRNA, and also the genes encoding small nucleolar RNAs
(snoRNAs) involved in rRNA processing and small nuclear RNAs (snRNAs) involved in mRNA
processing, except for U6 snRNA.

 RNA polymerase III (RNA Pol III) is also located in the nucleoplasm. It transcribes the genes
for tRNA, 5S rRNA, U6 snRNA, and the 7S RNA associated with the signal recognition particle
(SRP) involved in the translocation of proteins across the endoplasmic reticulum membrane.

 Each of the three eukaryotic RNA polymerases contains 12 or more subunits and so these are
large complex enzymes.

 The genes encoding some of the subunits of each eukaryotic enzyme show DNA sequence
similarities to genes encoding subunits of the core enzyme of E. coli RNA polymerase.

 However, four to seven other subunits of each eukaryotic RNA polymerase are unique in that
they show no similarity either with bacterial RNA polymerase subunits or with the subunits
of other eukaryotic RNA polymerases.

Features of eukaryotic transcription

 Eukaryotic Transcription occurs within the nucleus and mRNA moves out of the nucleus into
the cytoplasm for translation.

 The initiation of RNA synthesis by RNA polymerase is directed by the presence of a promoter
site on the 5’ side of the transcriptional start site.

 The RNA polymerase transcribes one strand, the antisense (-) strand, of the DNA template.

 RNA synthesis does not require a primer.

 RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a
nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on
an incoming ribonucleoside 5-triphosphate.

 mRNA in eukaryotes is processed from the primary RNA transcript, a process called
maturation.
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Process of Eukaryotic transcription

The basic mechanism of RNA synthesis by these eukaryotic RNA polymerases can be divided into the
following phases:

1. Initiation Phase

 During initiation, RNA polymerase recognizes a specific site on the DNA, upstream from the
gene that will be transcribed, called a promoter site and then unwinds the DNA locally.

 Most promoter sites for RNA polymerase II include a highly conserved sequence located
about 25–35 bp upstream (i.e. to the 5 side) of the start site which has the consensus
TATA(A/T)A(A/T) and is called the TATA box.

 Since the start site is denoted as position +1, the TATA box position is said to be located at
about position -25.

 The TATA box sequence resembles the -10 sequence in prokaryotes (TATAAT) except that it
is located further upstream.

 Both elements have essentially the same function, namely recognition by the RNA polymerase
in order to position the enzyme at the correct location to initiate transcription.

 The sequence around the TATA box is also important in that it influences the efficiency of
initiation. Transcription is also regulated by upstream control elements that lie 5′ to the TATA
box.

 Some eukaryotic protein-coding genes lack a TATA box and have an initiator element instead,
centered around the transcriptional initiation site.

 In order to initiate transcription, RNA polymerase II requires the assistance of several other
proteins or protein complexes, called general (or basal) transcription factors, which must
assemble into a complex on the promoter in order for RNA polymerase to bind and start
transcription.

 These all have the generic name of TFII (for Transcription Factor for RNA polymerase II).

 The first event in initiation is the binding of the transcription factor IID (TFIID) protein
complex to the TATA box via one its subunits called TBP (TATA box binding protein).

 As soon as the TFIID complex has bound, TFIIA binds and stabilizes the TFIID-TATA box
interaction. Next, TFIIB binds to TFIID.

 However, TFIIB can also bind to RNA polymerase II and so acts as a bridging protein. Thus,

 RNA polymerase II, which has already complexed with TFIIF, now binds.
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 This is followed by the binding of TFIIE and H. This final protein complex contains at least 40
polypeptides and is called the transcription initiation complex.

 Those protein-coding genes that have an initiator element instead of a TATA box appear to
need another protein(s) that binds to the initiator element.

 The other transcription factors then bind to form the transcription initiation comple

2. Elongation Phase

TFIIH has two functions:

1. It is a helicase, which means that it can use ATP to unwind the DNA helix, allowing
transcription to begin.

2. In addition, it phosphorylates RNA polymerase II which causes this enzyme to change its
conformation and dissociate from other proteins in the initiation complex.

 The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain (CTD)
of the RNA polymerase II molecule.

 Interestingly, only RNA polymerase II that has a non-phosphorylated CTD can initiate
transcription but only an RNA polymerase II with a phosphorylated CTD can elongate RNA.

 RNA polymerase II now starts moving along the DNA template, synthesizing RNA, that is, the
process enters the elongation phase.

 RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase catalyzing a
nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on
an incoming ribonucleoside 5-triphosphate.
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 The RNA molecule made from a protein-coding gene by RNA polymerase II is called a primary
transcript.

3. Termination Phase

 Elongation of the RNA chain continues until termination occurs.

 Unlike RNA polymerase in prokaryotes, RNA polymerase II does not terminate transcription
at a specific site but rather transcription can stop at varying distances downstream of the
gene.

 RNA genes transcribed by RNA Polymerse II lack any specific signals or sequences that direct
RNA Polymerase II to terminate at specific locations.

 RNA Polymerase II can continue to transcribe RNA anywhere from a few bp to thousands of
bp past the actual end of the gene.

 The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing.
This releases the upstream portion of the transcript, which will serve as the initial RNA prior
to further processing (the pre-mRNA in the case of protein-encoding genes.)

 This cleavage site is considered the “end” of the gene. The remainder of the transcript is
digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the
RNA Polymerase II.

 When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the
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overhanging RNA, it helps disengage the polymerase from its DNA template strand, finally
terminating that round of transcription.

RNA Processing

The primary eukaryotic mRNA transcript is much longer and localised into the nucleus, when it is also
called heterogenous nuclear RNA (hnRNA) or pre- mRNA.

It undergoes various processing steps to change into a mature RNA:

Cleavage

 Larger RNA precursors are cleaved to form smaller RNAs.

 Primary transcript is cleaved by ribonuclease-P (an RNA enzyme) to form 5-7 tRNA
precursors.

Capping and Tailing

 Initially at the 5′ end a cap (consisting of 7-methyl guanosine or 7 mG) and a tail of poly A at
the 3′ end are added.

 The cap is a chemically modified molecule of guanosine triphosphate (GTP).

Splicing

 The eukaryotic primary mRNAs are made up of two types of segments; non-coding introns
and the coding exons.

 The introns are removed by a process called RNA splicing where ATP is used to cut the RNA,
releasing the introns and joining two adjacent exons to produce mature mRNA.

Nucleotide Modifications
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 They are most common in tRNA-methylation (e.g., methyl cytosine, methyl guanosine),
deamination (e.g., inosine from adenine), dihydrouracil, pseudouracil, etc.

Characteristic of Genetic Code

 The genetic code is the set of rules by which a linear sequence of nucleotides specifies
the linear sequence of a polypeptide.

 That is, they specify how the nucleotide sequence of an mRNA is translated into
the amino acid sequence of a polypeptide.

 Thus, the relationship between the nucleotide sequence of the mRNA and the amino acid
sequence of the polypeptide is the genetic code.

 The nucleotide sequence is read as triplets called codons.

PRINCIPLES OF THE GENETIC CODE

 The genetic code consists of 64 different codons, each of which codes for 1 of the 20
amino acids.

 A codon consists of a triplet of nucleotide bases.

The genetic code is endowed with many characteristic properties which have actually been
proved by definite experimental evidences.

1. Triplet nature:

 Singlet and doublet codes are not adequate to code for 20 amino acids; therefore, it was
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pointed out that triplet code is the minimum required.

2. Degeneracy

 The code is degenerate which means that the same amino acid is coded by more than
one base triplet.

 Degeneracy does not imply lack of specificity in protein synthesis.

 It merely means that a particular amino acid can be directed to its place in the peptide
chain by more than one base triplets.

 For example, the three amino acids arginine, alanine and leucine each have six
synonymous codons.

 The code degeneracy is basically of 2 types: partial and complete.

 In partial degeneracy, the first two nucleotides are identical but the third (i.e., 3′ base)
nucleotide of the degenerate codon differs; for example, CUU and CUC code for leucine.

 Complete degeneracy occurs when any of the 4 bases can take third position and still
code for the same amino acid; for example, UCU, UCC, UCA and UCG all code for serine.

3. Non-overlapping

 The genetic code is nonoverlapping, i.e.,the adjacent codons do not overlap.

 A nonoverlapping code means that the same letter is not used for two different codons.
In other words, no single base can take part in the formation of more than one codon.

4. Commaless

 The genetic code is commaless (or comma-free). There is no signal to indicate the end of
one codon and the beginning of the next.

 There are no intermediary nucleotides (or commas) between the codons.

5. Non-ambiguity

 Non-ambiguous code means that there is no ambiguity about a particular codon.

 A particular codon will always code for the same amino acid.

 While the same amino acid can be coded by more than one codon (the code is
degenerate), the same codon shall not code for two or more different amino acids (non-
ambiguous).

6. Universality

 Universality of the code means that the same sequences of 3 bases encode the same
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amino acids in all life forms from simple microorganisms to complex, multicelled
organisms such as human beings.

 Although the code is based on work conducted on the bacterium Escherichia coli but it is
valid for other organisms.

 The genetic code applies to all modern organisms with only minor exceptions, such as
the yeast, mitochondria, and the Mycoplasma.

7. Polarity

 The genetic code has polarity, that is, the code is always read in a fixed direction, i.e., in
the 5′ → 3′ direction.

 It is apparent that if the code is read in opposite direction (i.e., 3′ → 5′), it would specify
2 different proteins, since the codon would have reversed base sequence.

Special Codons

A. Chain Initiation Codons

 The triplets AUG and GUG play double roles in E. coli.

 When they occur in between the two ends of a cistron (intermediate position), they code
for the amino acids methionine and valine, respectively in an intermediate position in
the protein molecule.

 But when they occur immediately after a terminator codon, they act as “chain initiation”
(C.I.) signals or “starter codons” for the synthesis of a polypeptide chain.

B. Chain Termination Codons

 The 3 triplets UAA, UAG, UGA do not code for any amino acid.

 When any one of them occurs immediately before the triplet AUG or GUG, it causes the
release of the polypeptide chain from the ribosome.

 They are also called as stop codons.

 They are also called chain termination codons because these codons are used by the cell
to signal the natural end of translation of a particular peptidyl chain. However, their
inclusion in any mRNA results in the abrupt termination of the message at the point of
their location even though the polypeptide chain has not been completed.

C. Sense Codons
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61 codons, which code for particular amino acids are termed as sense codons.

D. Non-Sense Codons

 Triplets UAA, UAG, UGA do not code for any amino acid.

 They were originally described as non-sense codons.

 However, the so-called non-sense codons have now been found to be of “special sense”.

 These special-sense codons perform the function of punctuating genetic message like a
full stop at the end of a sentence.

Prokaryotic – Translation

Translation involves translating the sequence of a messenger RNA (mRNA) molecule to a

sequence of amino acids during protein synthesis.

It is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the process
of transcription of DNA to RNA.

Ribosomes

 Ribosomes exist normally as separate subunits that are composed of protein and rRNA.

 The subunits come together to form a ribosome when they bind to an mRNA, near its 5’
end.

 On binding to an mRNA, the ribosome reads the nucleotide sequence from the 5’ to 3’
direction, synthesizing the corresponding protein from amino acids in an N-terminal
(amino-terminal) to C-terminal (carboxyl terminal) direction.

 Ribosomes are located in the cytosol, either freely floating or associated with the
endoplasmic reticulum.

 They serve to synthesize proteins.

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Ribosomal sites for Protein Translation

Each prokaryotic ribosome, shown schematically, has three binding sites for tRNAs.

1. The aminoacyl-tRNA binding site (or A site) is where, during elongation, the incoming
aminoacyl-tRNA binds.

2. The peptidyl-tRNA binding site (or P site) is where the tRNA linked to the growing
polypeptide chain is bound.

3. The exit site (or E site) is a binding site for tRNA following its role in translation and
prior to its release from the ribosome.

All three sites (A, P and E) are formed by the rRNA molecules in the ribosome

Translation Process

Protein synthesis (or translation) takes place in three stages:

1. Initiation

2. Elongation and

3. Termination.
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 During initiation, the mRNA–ribosome complex is formed and the first codon (always
AUG) binds the first aminoacyltRNA (called initiator tRNA).

 During the elongation phase, the other codons are read sequentially and the polypeptide
grows by addition of amino acids to its C-terminal end.

 This process continues until a termination codon (Stop codon), which does not have a
corresponding aminoacyl-tRNA with which to base pair, is reached.

 At this point, protein synthesis ceases (termination phase) and the finished polypeptide
is released from the ribosome.

Synthesis of amino-acyll tRNA

 Synthesis of aminoacyl-tRNAs is crucially important for two reasons:

1. Each amino acid must be covalently linked to a tRNA molecule in order to take part in
protein synthesis, which depends upon the ‘adaptor’ function of tRNA to ensure that the
correct amino acids are incorporated.

2. The covalent bond that is formed between the amino acid and the tRNA is a high energy
bond that enables the amino acid to react with the end of the growing polypeptide chain
to form a new peptide bond.

For this reason, the synthesis of aminoacyl-tRNA is also referred to as amino acid activation.

 Each tRNA molecule has a cloverleaf secondary structure with the anticodon accessible
at the end of the anticodon stem loop.

 During synthesis of the aminoacyl-tRNA, the amino acid is covalently bound to the A
residue of the CCA sequence at the 3’ end.

 Each tRNA molecule carries only a single amino acid.

 The attachment of an amino acid to a tRNA is catalyzed by an enzyme called aminoacyl-

tRNA synthetase.

 A separate aminoacyl-tRNA synthetase exists for every amino acid, making 20

synthetases in total.

The synthesis reaction occurs in two steps.

1. The first step is the reaction of an amino acid and ATP to form an aminoacyl-adenylate
(also known as aminoacyl-AMP).

2. In the second step, without leaving the enzyme, the aminoacyl group of aminoacyl-AMP
is transferred to the 3’ end of the tRNA molecule to form aminoacyl-tRNA
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The overall reaction is:

Amino acid + ATP + tRNA → aminoacyl-tRNA + AMP + PPi

Initiation of Protein synthesis

 The first codon translated in all mRNAs is the start codon or initiation codon, AUG which
codes for methionine.

 Two different tRNAs are used for the two types of AUG codon; tRNA fMet is used for the
initiation codon and is called the initiator tRNA whereas tRNA m Met is used for internal
AUG codons.

 In prokaryotes the first amino acid of a new protein is N-formylmethionine (abbreviated

fMet). Hence the aminoacyl-tRNA used in initiation is fMet-tRNAfMet.

 A short sequence rich in purines (5’-AGGAGGU-3’), called the Shine–Dalgarno

sequence, lies 5’ to the AUG initiation codon and is complementary to part of the 16S
rRNA in the small ribosomal subunit.

 Therefore this is the binding site for the 30S ribosomal subunit which then migrates in a
3’ direction along the mRNA until it encounters the AUG initiation codon.

 Initiation of protein synthesis requires proteins called initiation factors (IFs).

 In prokaryotes, three initiation factors (IF-1, IF-2 and IF-3) are essential.

 Because of the complexity of the process, the exact order of binding of IF-1, IF-2, IF-3,
fMet-tRNAf is controversial.

1. Initiation begins with the binding of IF-1 and IF-3 to the small (30S) ribosomal subunit.

 Their role is to stop the 30S subunit binding to the 50S subunit in the absence of mRNA
and fMet-tRNAf Met which would result in a nonfunctional ribosome.

2. The small subunit then binds to the mRNA via the Shine–Dalgarno sequence and moves
3’ along the mRNA until it locates the AUG initiation codon.

3. The initiator tRNA charged with N-formylmethionine and in a complex with IF-2 and GTP
(fMet-tRNAfMet/IF-2/GTP) now binds.

4. IF-3 is released.

5. The complex of mRNA, fMet-tRNAf Met, IF-1, IF-2 and the 30S ribosomal subunit is called
the 30S initiation complex.

6. The large (50S) ribosomal subunit now binds, with the release of IF-1 and IF-2 and
hydrolysis of GTP, to form a 70S initiation complex.
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Elongation of Protein Synthesis

 At the start of the first round of elongation, the initiation codon (AUG) is positioned in
the P site with fMet-tRNAfMet bound to it via codon–anticodon base pairing.

 The next codon in the mRNA is positioned in the A site.

 Elongation of the polypeptide chain occurs in three steps called the elongation cycle,
namely aminoacyl-tRNA binding, peptide bond formation and translocation:

Aminoacyl-tRNA binding

 The corresponding aminoacyl-tRNA for the second codon binds to the A site via codon–
anticodon interaction.

 Binding of the aminoacyl-tRNA requires elongation factor EF-Tu and GTP which bind as
an aminoacyl-tRNA/EF-Tu/GTP complex.

 Following binding, the GTP is hydrolyzed and the EF-Tu is released, now bound to GDP.

 Before the EF-Tu molecule can catalyze the binding of another charged tRNA to the
ribosome, it must be regenerated by a process involving another elongation factor, EF-
Ts.

This regeneration is called the EF-Tu–EF-Ts exchange cycle.

 First, EF-Ts binds to EF-Tu and displaces the GDP. Then GTP binds to the EF-Tu and
displaces EF-Ts. The EF-Tu-GTP is now ready to take part in another round of elongation.

Peptide bond formation

 The second step, peptide bond formation, is catalyzed by peptidyl transferase.

 In this reaction the carboxyl end of the amino acid bound to the tRNA in the P site is
uncoupled from the tRNA and becomes joined by a peptide bond to the amino group of
the amino acid linked to the tRNA in the A site.

Translocation

 In the third step, a complex of elongation factor EF-G (also called translocase) and GTP
(i.e. EF-G/GTP) binds to the ribosome.

 Three concerted movements now occur, collectively called translocation:

1. the deacylated tRNA moves from the P site to the E site

2. the dipeptidyl-tRNA in the A site moves to the P site, and

3. the ribosome moves along the mRNA (5’ to 3’) by three nucleotides to place the next
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codon in the A site.

 During the translocation events, GTP is hydrolyzed to GDP and inorganic phosphate, and
EF-G is released ready to bind more GTP for another round of elongation.

 After translocation, the A site is empty and ready to receive the next aminoacyltRNA.

 The A site and the E site cannot be occupied simultaneously. Thus the deacylated tRNA
is released from the E site before the next aminoacyl-tRNA binds to the A site to start a
new round of elongation.

 Elongation continues, adding one amino acid to the C-terminal end of the growing
polypeptide for each codon that is read, with the peptidyl-tRNA moving back and forth
from the P site to the A site as it grows.

Termination of Protein synthesis

 Eventually, one of three termination codons (also called Stop codons) becomes
positioned in the A site. These are UAG, UAA and UGA.

 Unlike other codons, prokaryotic cells do not contain aminoacyl-tRNAs complementary

to

 Stop codons. Instead, one of two release factors (RF-1 and RF-2) binds instead.

 RF-1 recognizes UAA and UAG whereas RF-2 recognizes UAA and UGA. A third release
factor, RF-3, is also needed to assist RF-1 or RF-2 interaction with the ribosome. Thus
either RF-1 + RF-3 or RF-2 + RF-3 bind depending on the exact termination codon in the
A site.

 RF-1 (or RF-2) binds at or near the A site whereas RF-3/GTP binds elsewhere on the
ribosome.

 The release factors cause the peptidyl transferase activity to transfer the polypeptide to
a water molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between
the polypeptide and tRNA in the P site.

The free polypeptide now leaves the ribosome, followed by the mRNA and free tRNA, and the
ribosome dissociates into 30S and 50S subunits ready to start translation again.

Eukaryotic Protein Synthesis

Protein Synthesis involves translating the sequence of a messenger RNA (mRNA) molecule to a
sequence of amino acids during protein synthesis. It is the process in which ribosomes in the
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cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA.

The Ribosomes

 Ribosomes exist normally as separate subunits that are composed of protein and rRNA.

 Eukaryotic ribosomes are larger (the 80S) and more complex than prokaryotic
ribosomes (70S).

 The subunits come together to form a ribosome when they bind to an mRNA, near its 5’
end.

 On binding to an mRNA, the ribosome reads the nucleotide sequence from the 5’ to 3’
direction, synthesizing the corresponding protein from amino acids in an N-terminal
(amino-terminal) to C-terminal (carboxyl-terminal) direction.

 Ribosomes are located in the cytosol, either freely floating or associated with the
endoplasmic reticulum.

 They serve to synthesize proteins.

Ribosomal sites for protein synthesis

Each prokaryotic ribosome, shown schematically, has three binding sites for tRNAs.

1. The aminoacyl-tRNA binding site (or A site) is where, during elongation, the incoming
aminoacyl-tRNA binds.

2. The peptidyl-tRNA binding site (or P site) is where the tRNA linked to the growing
polypeptide chain is bound.

3. The exit site (or E site) is a binding site for tRNA following its role in translation and prior
to its release from the ribosome.

All three sites (A, P, and E) are formed by the rRNA molecules in the ribosome.
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Protein synthesis process

 Whereas a prokaryotic ribosome has a sedimentation coefficient of the 70S and subunits
of 30S and 50S, a eukaryotic ribosome has a sedimentation coefficient of 80S with
subunits of 40S and 60S.

 The composition of eukaryotic ribosomal subunits is also more complex than

prokaryotic subunits but the function of each subunit is essentially the same as in
prokaryotes.

 In eukaryotes, each mRNA is monocistronic that is, discounting any subsequent post-
translational cleavage reactions that may occur; the mRNA encodes a single protein. In
prokaryotes, many mRNAs are polycistronic that is they encode several proteins. Each
coding sequence in a prokaryotic mRNA has its own initiation and termination codons.

 Initiation of protein synthesis in eukaryotes requires at least nine distinct eukaryotic

initiation factors (eIFs) compared with the three initiation factors (IFs) in prokaryotes.

 In eukaryotes, the initiating amino acid is methionine, not N-formylmethionine as in

prokaryotes.

 As in prokaryotes, a special initiator tRNA is required for initiation and is distinct from
the tRNA that recognizes and binds to codons for methionine at internal positions in the
mRNA. When charged with methionine ready to begin initiation, this is known as Met-
tRNAimet

 The main difference between initiation of translation in prokaryotes and eukaryotes is

that in bacteria, a Shine–Dalgarno sequence lies 5’ to the AUG initiation codon and is the
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binding site for the 30S ribosomal subunit.

 In contrast, most eukaryotic mRNAs do not contain Shine–Dalgarno sequences. Instead,

a 40S ribosomal subunit attaches at the 5’ end of the mRNA and moves downstream (i.e.
in a 5’ to 3’ direction) until it finds the AUG initiation codon. This process is called
scanning.

 Prokaryotic translation requires no helicase, presumably because protein synthesis in

bacteria can start even as the mRNA is still being synthesized whereas, in eukaryotes,
transcription in the nucleus and translation in the cytoplasm are separate events that
allow time for mRNA secondary structure to form.

Protein synthesis (or translation) takes place in three stages:

1. Initiation

2. Elongation and

3. Termination

Initiation of Protein synthesis

 The first step is the formation of a pre-initiation complex consisting of the 40S small
ribosomal subunit, Met-tRNAimet, eIF-2, and GTP.

 The pre-initiation complex binds to the 5’ end of the eukaryotic mRNA, a step that
requires eIF-4F (also called cap-binding complex) and eIF-3.

 The eIF-4F complex consists of eIF-4A, eIF-4E, and eIF-4G; eIF-4E binds to the 5’ cap on
the mRNA whilst eIF-4G interacts with the poly (A) binding protein on the poly (A) tail.

 The eIF-4A is an ATP-dependent RNA helicase that unwinds any secondary structures in
the mRNA, preparing it for translation.

 The complex then moves along the mRNA in a 5’ to 3’ direction until it locates the AUG
initiation codon (i.e. scanning).

 The 5’ untranslated regions of eukaryotic mRNAs vary in length but can be several
hundred nucleotides long and may contain secondary structures such as hairpin loops.
These secondary structures are probably removed by initiation factors of the scanning
complex.

 The initiation codon is usually recognizable because it is often (but not always)
contained in a short sequence called the Kozak consensus (5’-ACCAUGG-3’).

 Once the complex is positioned over the initiation codon, the 60S large ribosomal subunit
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binds to form an 80S initiation complex, a step that requires the hydrolysis of GTP and
leads to the release of several initiation factors.

Elongation of Protein Synthesis

 Elongation depends on eukaryotic elongation factors.

 Three elongation factors, eEF-1A, eEF-IB, and eEF-2, are involved which have similar
functions to their prokaryotic counterparts EF-Tu, EF-Ts and EF-G.

 At the end of the initiation step, the mRNA is positioned so that the next codon can be
translated during the elongation stage of protein synthesis.

 The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive
an aminoacyl-tRNA.

 During chain elongation, each additional amino acid is added to the nascent polypeptide
chain in a three-step microcycle.

 The steps in this microcycle are:

1. Positioning the correct aminoacyl-tRNA in the A site of the ribosome,

2. Forming the peptide bond and

3. Shifting the mRNA by one codon relative to the ribosome.

 Although most codons encode the same amino acids in both prokaryotes and eukaryotes,
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the mRNAs synthesized within the organelles of some eukaryotes use a variant of the
genetic code.

 During elongation in bacteria, the deacylated tRNA in the P site moves to the E site prior
to leaving the ribosome. In contrast, although the situation is still not completely clear,
in eukaryotes the deacylated tRNA appears to be ejected directly from the ribosome.

Termination of Protein synthesis

 Termination of elongation depends on eukaryotic release factors.

 In eukaryotes, eukaryotic release factor eRF-1 recognizes all three termination codons
(UAA, UAG, and UGA) and, with the help of protein eRF-3, terminates translation.

 Upon termination, the ribosome is disassembled and the completed polypeptide is

released
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