history molecular biology under the cell
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Sep 23, 2025
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About This Presentation
Landmarks in molecular biology through cell
Slide Content
QUESTION 1
A
Molecular Biology is the classic in-depth text reference in cell biology. By extracting
fundamental concepts and meaning from this enormous and ever-growing field, the story
of cell is told, and create a coherent framework through which non-expert readers may
approach the subject. Molecular Biology not only sets forth the current understanding of
cell biology, but also explores the intriguing implications and possibilities of that which
remains unknown
Cell
A structure containing a mass of cytoplasm surrounded by semi-permeable membrane
called plasma membrane is called a cell. It encloses cytoplasm, many cell organelles
along with nucleus or nuclear material. On the basis of organization of membranes,
variety and structure of cytoplasmic organelles and complexity of nuclear region, the
cells are classified into two types: Prokaryotic cell and Eukaryotic cell. These terms were
suggested by Hans Ris in 1960s.
Landmarks in molecular biology through cell
A cell was defined as “unit of biological activity delimited by a semi permeable
membrane and capable of self-reproduction in a medium free of other living systems” by
Loewy and Siekevitz (1963). The study of cell has been made possible with the help of
light microscope. Robert Hooke (1665) with the help of light microscope discovered that
a section of cork is made up of small cavities surrounded by firm walls. He used the term
“cell” for the first time to describe his investigations on the “texture of a piece of cork”.
Later on A. Van Leeuwenhoek (1632-1723) observed various unicellular organisms and
cells like bacteria, protozoan’s, red blood cells and sperm etc. He observed nucleus in
some erythrocytes and all this was made possible with the improved microscopes. In
1809, Mirble M. stated that all plant tissues are composed of cells. In the same year,
importance of cells in living organisms was described by J.B. Lamarck. Robert Brown in
1831 observed nucleus in certain plant cells. Mimosa cells were boiled in nitric acid by
Dutrochet (1837) to separate the cells to conclude that all organic tissues are composed of
globular cells, united by simple adhesive forces. “All living organism are composed of
cells” was stated by Schwann, T. (1839) after examining a variety of animals and plant
tissues.
It had been shown by Karl W. Nageli (1817-1891) that the cell membrane is
semipermeable and is responsible for the osmotic and other related phenomena exhibited
by living cells. Before 1855, he used the term zellen membrane in his early papers. The
term plasma membrane was used in 1855 by him to describe the membrane as a firm
protective film that is formed by out flowing cytoplasm of an injured cell when protein
rich cell sap came in contact with water.
Kölliker (1880) was the first who observed the mitochondria in insects muscle cells. He
called them as 'sarcosomes'. Flemming (1882) named the mitochondria as 'fila'. Altmann
A
Molecular Biology is the classic in-depth text reference in cell biology. By extracting
fundamental concepts and meaning from this enormous and ever-growing field, the story
of cell is told, and create a coherent framework through which non-expert readers may
approach the subject. Molecular Biology not only sets forth the current understanding of
cell biology, but also explores the intriguing implications and possibilities of that which
remains unknown
Cell
A structure containing a mass of cytoplasm surrounded by semi-permeable membrane
called plasma membrane is called a cell. It encloses cytoplasm, many cell organelles
along with nucleus or nuclear material. On the basis of organization of membranes,
variety and structure of cytoplasmic organelles and complexity of nuclear region, the
cells are classified into two types: Prokaryotic cell and Eukaryotic cell. These terms were
suggested by Hans Ris in 1960s.
Landmarks in molecular biology through cell
A cell was defined as “unit of biological activity delimited by a semi permeable
membrane and capable of self-reproduction in a medium free of other living systems” by
Loewy and Siekevitz (1963). The study of cell has been made possible with the help of
light microscope. Robert Hooke (1665) with the help of light microscope discovered that
a section of cork is made up of small cavities surrounded by firm walls. He used the term
“cell” for the first time to describe his investigations on the “texture of a piece of cork”.
Later on A. Van Leeuwenhoek (1632-1723) observed various unicellular organisms and
cells like bacteria, protozoan’s, red blood cells and sperm etc. He observed nucleus in
some erythrocytes and all this was made possible with the improved microscopes. In
1809, Mirble M. stated that all plant tissues are composed of cells. In the same year,
importance of cells in living organisms was described by J.B. Lamarck. Robert Brown in
1831 observed nucleus in certain plant cells. Mimosa cells were boiled in nitric acid by
Dutrochet (1837) to separate the cells to conclude that all organic tissues are composed of
globular cells, united by simple adhesive forces. “All living organism are composed of
cells” was stated by Schwann, T. (1839) after examining a variety of animals and plant
tissues.
It had been shown by Karl W. Nageli (1817-1891) that the cell membrane is
semipermeable and is responsible for the osmotic and other related phenomena exhibited
by living cells. Before 1855, he used the term zellen membrane in his early papers. The
term plasma membrane was used in 1855 by him to describe the membrane as a firm
protective film that is formed by out flowing cytoplasm of an injured cell when protein
rich cell sap came in contact with water.
Kölliker (1880) was the first who observed the mitochondria in insects muscle cells. He
called them as 'sarcosomes'. Flemming (1882) named the mitochondria as 'fila'. Altmann
in 1894 observed them and named them Altmann's granules or bioblasts. The term
'mitochondria' was applied by Benda (1897-98). They were recognized as the sites of
respiration by Hogeboom and his coworkers in 1948. Lehninger and Kennedy (1948)
reported that the mitochondria catalyze all the reactions of the citric acid cycle, fatty acid
oxidation and coupled phosphorylation.
Early cytologists held that some sort of supporting network or cytoskeleton was present
in the cells. It was given various names — Nissil substance, ergastoplasm, basophilic
bodies, etc. In 1945, Porter, Claude and Fullman with the help of electron microscope
noted a delicate membranous network in the cytoplasm. It was later called endoplasmic
reticulum (ER) by Keith Porter in 1953. The ER originally seemed to be confined to the
endoplasm of the cell, hence its name.
George E. Palade (1953) was the first to observe dense particles or granules in animal
cells under electron microscope. These were thus called as Palade’s Particles. Later
Richard B. Roberts named them "ribosomes" in 1958. Tissieres and J.D. Watson (1958)
isolated ribosomes from E. coli for the first time. It was shown that ribosomes contain
approximately equal amount of RNA and proteins.
Camillo Golgi in 1898 discovered the Golgi apparatus in the nerve cells of barn owl and
cat by metallic impregnation method. After it's discoverer's name, the Golgi apparatus
has been variously named as Golgisome, Golgi material, Golgi membranes, Golgi body,
etc.
Lysosome is an organelle which unlike other organelles, first became known through the
biochemical studies and thereafter their morphological identifications were made.
Christian de Duve, a Belgian cytologist and biochemist, in 1955 reported the presence of
lysosomes in the cells by biochemical studies. Later on, Novikoff in 1956 observed these
lysosomes as distinct cell organelles with the help of electron microscope.
The cytologists like Freud (1882), Ballowitz (1890) and Meves (1910) observed
filamentous components of the cytoplasm and referred these as fibrils. Later, with the
improved microscopic techniques along with advancement made in the field of sectioning
and staining, the ultra structure of these components was revealed. These were found to
be tubular in nature (Burgos and Fawcett, 1955; Palay, 1960; Harris, 1962). De Robertis
and Franchi (1953) reported the presence of microtubules in the axons of medullated
nerve fibers and called them neurotubules. Slautterback in 1963 describes them to be
associated with the developing nematocysts of Hydra and he proposed the name
microtubules to these components.
Nucleus was observed by a Dutch Microscopist, Antonie van Leeuwenhoek in 1710, as a
centrally placed clear area in the blood cells of amphibians and birds. Fontana (1781)
recorded an ovoid structure in each of the isolated epidermal cells of eel's skin. However,
Robert Brown (1831) was the first to use the term nucleus for a prominent body present
'mitochondria' was applied by Benda (1897-98). They were recognized as the sites of
respiration by Hogeboom and his coworkers in 1948. Lehninger and Kennedy (1948)
reported that the mitochondria catalyze all the reactions of the citric acid cycle, fatty acid
oxidation and coupled phosphorylation.
Early cytologists held that some sort of supporting network or cytoskeleton was present
in the cells. It was given various names — Nissil substance, ergastoplasm, basophilic
bodies, etc. In 1945, Porter, Claude and Fullman with the help of electron microscope
noted a delicate membranous network in the cytoplasm. It was later called endoplasmic
reticulum (ER) by Keith Porter in 1953. The ER originally seemed to be confined to the
endoplasm of the cell, hence its name.
George E. Palade (1953) was the first to observe dense particles or granules in animal
cells under electron microscope. These were thus called as Palade’s Particles. Later
Richard B. Roberts named them "ribosomes" in 1958. Tissieres and J.D. Watson (1958)
isolated ribosomes from E. coli for the first time. It was shown that ribosomes contain
approximately equal amount of RNA and proteins.
Camillo Golgi in 1898 discovered the Golgi apparatus in the nerve cells of barn owl and
cat by metallic impregnation method. After it's discoverer's name, the Golgi apparatus
has been variously named as Golgisome, Golgi material, Golgi membranes, Golgi body,
etc.
Lysosome is an organelle which unlike other organelles, first became known through the
biochemical studies and thereafter their morphological identifications were made.
Christian de Duve, a Belgian cytologist and biochemist, in 1955 reported the presence of
lysosomes in the cells by biochemical studies. Later on, Novikoff in 1956 observed these
lysosomes as distinct cell organelles with the help of electron microscope.
The cytologists like Freud (1882), Ballowitz (1890) and Meves (1910) observed
filamentous components of the cytoplasm and referred these as fibrils. Later, with the
improved microscopic techniques along with advancement made in the field of sectioning
and staining, the ultra structure of these components was revealed. These were found to
be tubular in nature (Burgos and Fawcett, 1955; Palay, 1960; Harris, 1962). De Robertis
and Franchi (1953) reported the presence of microtubules in the axons of medullated
nerve fibers and called them neurotubules. Slautterback in 1963 describes them to be
associated with the developing nematocysts of Hydra and he proposed the name
microtubules to these components.
Nucleus was observed by a Dutch Microscopist, Antonie van Leeuwenhoek in 1710, as a
centrally placed clear area in the blood cells of amphibians and birds. Fontana (1781)
recorded an ovoid structure in each of the isolated epidermal cells of eel's skin. However,
Robert Brown (1831) was the first to use the term nucleus for a prominent body present
in the orchid cell. He stated that nucleus was the regular feature of the cells and initiated
the concept of nucleated cells.
W. Hofmeister in 1848, discovered nuclear filaments in the nuclei of pollen mother cells
of Tradescantia. First accurate count of chromosomes was made by W. Flemming in
1882, in the nucleus of a cell. In 1884, W. Flemming, Evan Beneden and E. Strasburger
demonstrated that the chromosomes double in number by longitudinal division during
mitosis. Beneden in 1887 found that the number of chromosomes for each species was
constant. The term "Chromosomes" was coined in 1888 by W. Waldeyer for the nuclear
filaments. W.S. Sutton and T. Boveri suggested the role of chromosomes in heredity in
1902, which was confirmed by Morgan in 1933.
Structure of the cell
A cell
Types of cell
1. Prokaryotic cells are the most primitive cells and have simple structural organization.
It has a single membrane system. They include bacteria, viruses, blue-green algae,
mycoplasmas, rickettsias, spirochetes etc. Cyanobacteria or blue green algae are the
largest and most complex prokaryote, in which photosynthesis of higher plants type have
evolved. Prokaryotes are included in the kingdom Monera and the super kingdom
Prokaryota. The Prokaryotes have the following characters:
1. The size of prokaryotic cells ranges between 1 to 10 µm. They occur in a variety of
forms.
the concept of nucleated cells.
W. Hofmeister in 1848, discovered nuclear filaments in the nuclei of pollen mother cells
of Tradescantia. First accurate count of chromosomes was made by W. Flemming in
1882, in the nucleus of a cell. In 1884, W. Flemming, Evan Beneden and E. Strasburger
demonstrated that the chromosomes double in number by longitudinal division during
mitosis. Beneden in 1887 found that the number of chromosomes for each species was
constant. The term "Chromosomes" was coined in 1888 by W. Waldeyer for the nuclear
filaments. W.S. Sutton and T. Boveri suggested the role of chromosomes in heredity in
1902, which was confirmed by Morgan in 1933.
Structure of the cell
A cell
Types of cell
1. Prokaryotic cells are the most primitive cells and have simple structural organization.
It has a single membrane system. They include bacteria, viruses, blue-green algae,
mycoplasmas, rickettsias, spirochetes etc. Cyanobacteria or blue green algae are the
largest and most complex prokaryote, in which photosynthesis of higher plants type have
evolved. Prokaryotes are included in the kingdom Monera and the super kingdom
Prokaryota. The Prokaryotes have the following characters:
1. The size of prokaryotic cells ranges between 1 to 10 µm. They occur in a variety of
forms.
2. Prokaryotic cell consists of three main components: (I) Outer covering: It is composed
of inner cell or plasma membrane, middle cell wall and outer slimy capsule. a. 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.
b. 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 composed of
peptidoglycans. . Some bacteria such as mycoplasmas lack cell wall.
c. 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
(II) 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:
a. It lacks cell organelles like endoplasmic reticulum, mitochondria, Golgi apparatus,
Centrosomes, vacuoles, Lysosomes, microfilaments, intermediate filaments and
microtubules.
b. 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.
c. Like eukaryotic cells, the cytoplasm of prokaryotic cell does not show streaming
movement or cyclosis.
d. Gas vacuoles are also formed in some prokaryotic cells.
e. The cell does not show phagocytosis, pinocytosis and exocytose, substances enter and
leave the cell through the cell membrane.
f. They may contain deposits of polysaccharides or inorganic phosphates.
(III) 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. A short and simple pro-chromosome is present which is attached at least at one point
on cell membrane.
b. Mostly there is single copy of chromosome, the prokaryotic cell is haploid.
c. The DNA is naked as it is not associated with basic histone proteins. It is double
stranded, helical and circular.
of inner cell or plasma membrane, middle cell wall and outer slimy capsule. a. 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.
b. 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 composed of
peptidoglycans. . Some bacteria such as mycoplasmas lack cell wall.
c. 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
(II) 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:
a. It lacks cell organelles like endoplasmic reticulum, mitochondria, Golgi apparatus,
Centrosomes, vacuoles, Lysosomes, microfilaments, intermediate filaments and
microtubules.
b. 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.
c. Like eukaryotic cells, the cytoplasm of prokaryotic cell does not show streaming
movement or cyclosis.
d. Gas vacuoles are also formed in some prokaryotic cells.
e. The cell does not show phagocytosis, pinocytosis and exocytose, substances enter and
leave the cell through the cell membrane.
f. They may contain deposits of polysaccharides or inorganic phosphates.
(III) 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. A short and simple pro-chromosome is present which is attached at least at one point
on cell membrane.
b. Mostly there is single copy of chromosome, the prokaryotic cell is haploid.
c. The DNA is naked as it is not associated with basic histone proteins. It is double
stranded, helical and circular.
d. 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.
e. The processes like meiosis, gamete formation or fertilization are absent. Conjugation is
seen in some bacteria.
f. Mitotic apparatus absent.
g. There is no nucleolus.
h. Cell membrane folds or mesosomes help to segregate the replicated products of
chromosomes into daughter cells.
3. 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 to 30,000
base pairs and they generally encode proteins required by the organism to resist antibiotic
and other toxic material.
4. 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.
(i) 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.
(ii) 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.
5. 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
synthetic activities than eukaryotes.
2. Eukaryotic Cells
The internal organization of eukaryotic cell is more developed than prokaryotic cells
from which they are believed to have been evolved. They are evolved to have double
membrane system. Primary membranes are the one that surrounds the cell, celled cell or
plasma membrane and the secondary membrane surround the nucleus and other cellular
organelles. Eukaryotic cells occur in protists, fungi, plants and animals. Eukaryotic cells
have the following characteristics:
1. Number- In multicellular organisms the numbers of cells are correlated with the body
size. The human blood contains about 30 quadrillion (3 × 1015) corpuscles and a 60 kg
human being has about 60 × 1015 cells. All multicellular organisms begin their life with a
single cell “Zygote” and then become multicellular by its mitotic division during
development.
Replication of DNA is continuous throughout the cell cycle. Transcription and translation
occurs in cytoplasm and processing of mRNA is not required.
e. The processes like meiosis, gamete formation or fertilization are absent. Conjugation is
seen in some bacteria.
f. Mitotic apparatus absent.
g. There is no nucleolus.
h. Cell membrane folds or mesosomes help to segregate the replicated products of
chromosomes into daughter cells.
3. 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 to 30,000
base pairs and they generally encode proteins required by the organism to resist antibiotic
and other toxic material.
4. 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.
(i) 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.
(ii) 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.
5. 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
synthetic activities than eukaryotes.
2. Eukaryotic Cells
The internal organization of eukaryotic cell is more developed than prokaryotic cells
from which they are believed to have been evolved. They are evolved to have double
membrane system. Primary membranes are the one that surrounds the cell, celled cell or
plasma membrane and the secondary membrane surround the nucleus and other cellular
organelles. Eukaryotic cells occur in protists, fungi, plants and animals. Eukaryotic cells
have the following characteristics:
1. Number- In multicellular organisms the numbers of cells are correlated with the body
size. The human blood contains about 30 quadrillion (3 × 1015) corpuscles and a 60 kg
human being has about 60 × 1015 cells. All multicellular organisms begin their life with a
single cell “Zygote” and then become multicellular by its mitotic division during
development.
2. Shape- A cell may be spherical, cuboidal, oval, disc-like, polygonal, columnar, spindle
like or irregular. Thus, cells acquire a variety of shapes not only in various organisms but
also in different tissues of the same organism. The shape of cell is correlated with its
functions like the shape of muscles and nerve cells are well adapted to their functions.
Many factors such as cell functions, age of cell, presence or absence of cell wall,
viscosity of cytoplasm etc. are responsible for various shapes of cells.
3. Size- Most of the eukaryotic cells is microscopic and their size ranges between 10 to
100µm. Sporozoits of malaria parasite (Plasmodium vivax) is among the smallest cells
having the size equal to 2µm long. While the Ostrich egg measures 175 × 120mm. Nerve
cells are the longest having the size of its fiber to be of few meters long. Human cells
generally range from 20 to 30µm.
4. Components of a cell- Three main components of the eukaryotic cells are cell
membrane, cytoplasm and nucleus. The cytoplasm and the nucleus further have several
components. Various cell components are discussed below:
(i) Cell membrane- Cell membrane, plasma membrane or plasmalemma is a thin elastic
living covering that surrounds the cell keeping the cell contents in place, provides shape
to the cell and controls the transfer of materials across it. It is composed of lipid-protein
complex. It lacks respiratory enzymes. In many protists and animal cells it allows
endocytosis and exocytosis. In certain protists, many fungi and all plant cells, the cell
membrane is covered by a thick, rigid non-living cell wall that protects and supports the
cell. In prokaryotes the cell wall surrounding the plasma membrane has a different
structure in comparison to eukaryotes.
(ii) Cytoplasm- The cytoplasm or the cytosome is a semi-fluid, homogeneous, translucent
ground substance known as cytoplasmic matrix or cytosol which is present between the
cell membrane and the nucleus. In the protozoan cell the outer firm layer of cytoplasm is
called ectoplasm and the inner layer around the central fluid mass is called the
endoplasm. The cytosol shows “cyclosis” or the streaming movement. The eukaryotic
cytoplasm has the following features:-
a. Organelles: The organized structures having the specific functions and capacity of
growth and multiplication in some cases are known as organelles. Mitochondria,
centrosomes, Golgi bodies, plastids and vacuoles are the organelles that can be observed
under light microscope, while endoplasmic reticulum, ribosome, microfilaments,
microtubules, intermediate filaments and micro bodies can only be seen under electron
microscope. These organelles are often described as protoplasmic structures. The cells
having cilia or flagella have their basal bodies at the bases are in the cytoplasm while rest
of its part extends out of cytoplasm. These organelles are described as follows:
I. Mitochondria: The rod like or globule shaped structures scattered in the cytoplasm are
found singly or in groups. They are bounded by double membrane of lipoproteins. The
inner membrane gives out finger like structure known as cristae which partially subdivide
the inner chamber of mitochondrion. On the inner surface of cristae are present
mushroom like structures, oxysomes that are related to phosphorylation. The space
between the membranes and its lumen is filled with mitochondrial matrix. Both the
membranes and the matrix contain many oxidative enzymes and coenzymes. Since
mitochondria contain DNA molecules and ribosomes, they synthesize certain proteins.
like or irregular. Thus, cells acquire a variety of shapes not only in various organisms but
also in different tissues of the same organism. The shape of cell is correlated with its
functions like the shape of muscles and nerve cells are well adapted to their functions.
Many factors such as cell functions, age of cell, presence or absence of cell wall,
viscosity of cytoplasm etc. are responsible for various shapes of cells.
3. Size- Most of the eukaryotic cells is microscopic and their size ranges between 10 to
100µm. Sporozoits of malaria parasite (Plasmodium vivax) is among the smallest cells
having the size equal to 2µm long. While the Ostrich egg measures 175 × 120mm. Nerve
cells are the longest having the size of its fiber to be of few meters long. Human cells
generally range from 20 to 30µm.
4. Components of a cell- Three main components of the eukaryotic cells are cell
membrane, cytoplasm and nucleus. The cytoplasm and the nucleus further have several
components. Various cell components are discussed below:
(i) Cell membrane- Cell membrane, plasma membrane or plasmalemma is a thin elastic
living covering that surrounds the cell keeping the cell contents in place, provides shape
to the cell and controls the transfer of materials across it. It is composed of lipid-protein
complex. It lacks respiratory enzymes. In many protists and animal cells it allows
endocytosis and exocytosis. In certain protists, many fungi and all plant cells, the cell
membrane is covered by a thick, rigid non-living cell wall that protects and supports the
cell. In prokaryotes the cell wall surrounding the plasma membrane has a different
structure in comparison to eukaryotes.
(ii) Cytoplasm- The cytoplasm or the cytosome is a semi-fluid, homogeneous, translucent
ground substance known as cytoplasmic matrix or cytosol which is present between the
cell membrane and the nucleus. In the protozoan cell the outer firm layer of cytoplasm is
called ectoplasm and the inner layer around the central fluid mass is called the
endoplasm. The cytosol shows “cyclosis” or the streaming movement. The eukaryotic
cytoplasm has the following features:-
a. Organelles: The organized structures having the specific functions and capacity of
growth and multiplication in some cases are known as organelles. Mitochondria,
centrosomes, Golgi bodies, plastids and vacuoles are the organelles that can be observed
under light microscope, while endoplasmic reticulum, ribosome, microfilaments,
microtubules, intermediate filaments and micro bodies can only be seen under electron
microscope. These organelles are often described as protoplasmic structures. The cells
having cilia or flagella have their basal bodies at the bases are in the cytoplasm while rest
of its part extends out of cytoplasm. These organelles are described as follows:
I. Mitochondria: The rod like or globule shaped structures scattered in the cytoplasm are
found singly or in groups. They are bounded by double membrane of lipoproteins. The
inner membrane gives out finger like structure known as cristae which partially subdivide
the inner chamber of mitochondrion. On the inner surface of cristae are present
mushroom like structures, oxysomes that are related to phosphorylation. The space
between the membranes and its lumen is filled with mitochondrial matrix. Both the
membranes and the matrix contain many oxidative enzymes and coenzymes. Since
mitochondria contain DNA molecules and ribosomes, they synthesize certain proteins.
They produce the energy and reserve it in the form of adenosine triphosphate (ATP). Due
to the presence of its own DNA and ability of protein synthesis along with its duplication,
the mitochondria are called semi autonomous organelle. The DNA of mitochondria
resembles that of bacterial cell; hence it is also called as endo-symbiotic organelle.
II. Centrosomes: (9+0) there is a clear zone around centrioles, near the nucleus, that
includes a specialized portion of cytoplasm, called centrospheres. Its matrix is called
kinoplasm that bears two rounded bodies the “centrioles”. Each centriole consists of nine
fibrillar units and each of them is found to contain three microtubules arranged in a circle.
Both the centrioles are arranged at right angle to each other. Centrioles form the spindles
of microtubules at the time of cell division. Centrioles are absent in plant cell and the
spindle is formed without their help.
III. Golgi bodies: These are the stack of flattened parallel-arranged sacs and vesicles
found in association of endoplasmic reticulum. They are composed of many lamellae,
tubules, vesicles and vacuoles. Their membranes are supposed to be originated from ER
and are composed of lipoproteins. In plant cells the Golgi complex is called dictyosome
that secretes required materials for the formation of cell wall at the time of cell division.
It helps in the formation of acrosome of sperms, release of hormones, enzymes and other
synthetic materials.
IV. Plastids: These organelles are found in plant cells and are absent in animal cells. They
may be colored like chloroplast or chromoplasts or colorless like leucoplast. Since the
leucoplast store and metabolise the starch and lipids, they are called amyloplast and
lipoplast respectively. Chloroplast contains the green pigment the chlorophyll that helps
in photosynthesis and protein storage. Chloroplast has a double outer membrane, the
stroma, that bears many soluble enzymes, and a complex system of membrane bound
compartments called thalakoids constituting granna. Like mitochondria, chloroplast also
has their own DNA, ribosomes and complete protein synthetic machinery. Hence these
are also called endo-symbiotic and semi-autonomous organelle. V. Metaplasm: The
particles like vacuoles, granules and other cytoplasmic bodies such as ribonucleoprotein
molecules are represented by it.
VI. Cilia, basal bodies and flagella: Cilia are the minute structures covering the surface in
some cells. Both cilia and flagella originate from the basal bodies or blepharoplast lying
in cytoplasm. They consist of nine outer fibrils with the two larger fibrils in the centre.
Each fibril consists of two microtubules, or has 9+2 arrangement. Cilia and Flagella are
the structure born by certain cells. They are composed of microtubules made of the
protein tubulin. They have 9 + 2 plan of microtubule. Both grow at the base. They act as
locomotory organelles, moves by their beats or undulations for they get the energy by
breakdown of ATP molecule.
VII. Microtubules: The ultra fine tubules of protein (tubulin) traversing the cytoplasm of
plant and animal cells providing the structural framework to the cell, determine the cell
shape and general organization of the cytoplasm are known as microtubules. Tubules are
made up of 13 individual filaments. Microtubules help in transport of water and ions,
cytoplasmic streaming (cyclosis) and the formation of spindles during cell division.
VIII. Basal granules: The spherical bodies found at the base of cilia and flagella are
called the basal bodies. Each of them is composed of nine fibrils and each fibril consists
of the three microtubules, out of which two enter the cilia or flagella.
to the presence of its own DNA and ability of protein synthesis along with its duplication,
the mitochondria are called semi autonomous organelle. The DNA of mitochondria
resembles that of bacterial cell; hence it is also called as endo-symbiotic organelle.
II. Centrosomes: (9+0) there is a clear zone around centrioles, near the nucleus, that
includes a specialized portion of cytoplasm, called centrospheres. Its matrix is called
kinoplasm that bears two rounded bodies the “centrioles”. Each centriole consists of nine
fibrillar units and each of them is found to contain three microtubules arranged in a circle.
Both the centrioles are arranged at right angle to each other. Centrioles form the spindles
of microtubules at the time of cell division. Centrioles are absent in plant cell and the
spindle is formed without their help.
III. Golgi bodies: These are the stack of flattened parallel-arranged sacs and vesicles
found in association of endoplasmic reticulum. They are composed of many lamellae,
tubules, vesicles and vacuoles. Their membranes are supposed to be originated from ER
and are composed of lipoproteins. In plant cells the Golgi complex is called dictyosome
that secretes required materials for the formation of cell wall at the time of cell division.
It helps in the formation of acrosome of sperms, release of hormones, enzymes and other
synthetic materials.
IV. Plastids: These organelles are found in plant cells and are absent in animal cells. They
may be colored like chloroplast or chromoplasts or colorless like leucoplast. Since the
leucoplast store and metabolise the starch and lipids, they are called amyloplast and
lipoplast respectively. Chloroplast contains the green pigment the chlorophyll that helps
in photosynthesis and protein storage. Chloroplast has a double outer membrane, the
stroma, that bears many soluble enzymes, and a complex system of membrane bound
compartments called thalakoids constituting granna. Like mitochondria, chloroplast also
has their own DNA, ribosomes and complete protein synthetic machinery. Hence these
are also called endo-symbiotic and semi-autonomous organelle. V. Metaplasm: The
particles like vacuoles, granules and other cytoplasmic bodies such as ribonucleoprotein
molecules are represented by it.
VI. Cilia, basal bodies and flagella: Cilia are the minute structures covering the surface in
some cells. Both cilia and flagella originate from the basal bodies or blepharoplast lying
in cytoplasm. They consist of nine outer fibrils with the two larger fibrils in the centre.
Each fibril consists of two microtubules, or has 9+2 arrangement. Cilia and Flagella are
the structure born by certain cells. They are composed of microtubules made of the
protein tubulin. They have 9 + 2 plan of microtubule. Both grow at the base. They act as
locomotory organelles, moves by their beats or undulations for they get the energy by
breakdown of ATP molecule.
VII. Microtubules: The ultra fine tubules of protein (tubulin) traversing the cytoplasm of
plant and animal cells providing the structural framework to the cell, determine the cell
shape and general organization of the cytoplasm are known as microtubules. Tubules are
made up of 13 individual filaments. Microtubules help in transport of water and ions,
cytoplasmic streaming (cyclosis) and the formation of spindles during cell division.
VIII. Basal granules: The spherical bodies found at the base of cilia and flagella are
called the basal bodies. Each of them is composed of nine fibrils and each fibril consists
of the three microtubules, out of which two enter the cilia or flagella.
IX. Ribosome’s: Ribosome is the minute spherical structures that originate in nucleolus
and are found attached with the membrane of endoplasmic reticulum and in the
cytoplasm. They are mainly composed of ribonucleic acids (RNA) and protein. They are
mainly responsible for protein synthesis. b. Inclusions: These are the non-living or
deutoplasmic structures which are incapable of growth and multiplication. Common cell
inclusions are stored organic materials such as starch grains, glycogen granules, aleuron
grains, fat droplets, pigment granules and inorganic crystals.Cytoplasm is stores raw
materials needed for the metabolism in both the cytoplasm and the nucleus. Many
metabolic processes like biosynthesis of fatty acids, nucleotides, proteins and oxidation
take place in cytoplasm. It distributes the nutrients, metabolites and enzymes in a cell and
brings about exchange of materials between the organelles as well as with the
environment or extracellular fluid also.
c. Nucleus: In a eukaryotic cell the genetic material is enclosed by a distinct nuclear
envelope that forms a prominent spherical organelle the “Nucleus”. The nuclear envelope
bears pores for the exchange of materials between the cytoplasm and the nucleoplasm.
SUMMARY
Robert Hook (1665) for the first time described the texture of a piece of cork as “cell”.
Similar structures were observed by many scientists while studying many living
organisms. It was Schwann T. (1839) who stated that all living organisms are composed
of cells after examining a variety of plant and animal tissues. Basically two types of cells
are there, “Prokaryotic” and “Eukaryotic”. Prokaryotic cells are the primitive cells that
include bacteria, blue-green algae, viruses and photosynthetic cells cyanobacteria etc.
Their size varies from 1 to 10 um and they consist of mainly three components: the outer
covering that includes all cell membrane, cell wall and a slimy capsule. Another
component is cytoplasm which lacks cell organelles except ribosomes. The processes like
phagocytosis and endocytosis are absent. The third component is nucleoid that lacks
nuclear membrane. Additional small circular DNA the plasmid may also be present.
Flagella and pili like structure are also seen in some prokaryotic cells. Eukaryotic cells
are more developed and are surrounded by double membranes. Shape and size of these
cells and their number in multicellular organisms varies. It is also composed of three
main components. Cell membrane or plasma membrane is a thin elastic living covering.
The cytoplasm is a semi fluid, homogenous, translucent consisting of many cell
organelles, inclusions, cilia, flagella, basal bodies and microtubules.
Reference
Brown, R. (1831). Observations on the organs and mode of fecundation in Orchideae and
Asclepiadeae. Trans. Linn. Soc. London, 16: 685-746.
Dutrochet, H. (1837). Memoires pour servir á l’ histoire anatomique et physiologique des
végétaux et des animaux. Bailliere, Paris.
Hooke, R. (1665). Micrographia: or some physiological descriptions of minute bodies
made by magnifying glasses with observations and inquiries thereupon. Royal Society,
London, UK
and are found attached with the membrane of endoplasmic reticulum and in the
cytoplasm. They are mainly composed of ribonucleic acids (RNA) and protein. They are
mainly responsible for protein synthesis. b. Inclusions: These are the non-living or
deutoplasmic structures which are incapable of growth and multiplication. Common cell
inclusions are stored organic materials such as starch grains, glycogen granules, aleuron
grains, fat droplets, pigment granules and inorganic crystals.Cytoplasm is stores raw
materials needed for the metabolism in both the cytoplasm and the nucleus. Many
metabolic processes like biosynthesis of fatty acids, nucleotides, proteins and oxidation
take place in cytoplasm. It distributes the nutrients, metabolites and enzymes in a cell and
brings about exchange of materials between the organelles as well as with the
environment or extracellular fluid also.
c. Nucleus: In a eukaryotic cell the genetic material is enclosed by a distinct nuclear
envelope that forms a prominent spherical organelle the “Nucleus”. The nuclear envelope
bears pores for the exchange of materials between the cytoplasm and the nucleoplasm.
SUMMARY
Robert Hook (1665) for the first time described the texture of a piece of cork as “cell”.
Similar structures were observed by many scientists while studying many living
organisms. It was Schwann T. (1839) who stated that all living organisms are composed
of cells after examining a variety of plant and animal tissues. Basically two types of cells
are there, “Prokaryotic” and “Eukaryotic”. Prokaryotic cells are the primitive cells that
include bacteria, blue-green algae, viruses and photosynthetic cells cyanobacteria etc.
Their size varies from 1 to 10 um and they consist of mainly three components: the outer
covering that includes all cell membrane, cell wall and a slimy capsule. Another
component is cytoplasm which lacks cell organelles except ribosomes. The processes like
phagocytosis and endocytosis are absent. The third component is nucleoid that lacks
nuclear membrane. Additional small circular DNA the plasmid may also be present.
Flagella and pili like structure are also seen in some prokaryotic cells. Eukaryotic cells
are more developed and are surrounded by double membranes. Shape and size of these
cells and their number in multicellular organisms varies. It is also composed of three
main components. Cell membrane or plasma membrane is a thin elastic living covering.
The cytoplasm is a semi fluid, homogenous, translucent consisting of many cell
organelles, inclusions, cilia, flagella, basal bodies and microtubules.
Reference
Brown, R. (1831). Observations on the organs and mode of fecundation in Orchideae and
Asclepiadeae. Trans. Linn. Soc. London, 16: 685-746.
Dutrochet, H. (1837). Memoires pour servir á l’ histoire anatomique et physiologique des
végétaux et des animaux. Bailliere, Paris.
Hooke, R. (1665). Micrographia: or some physiological descriptions of minute bodies
made by magnifying glasses with observations and inquiries thereupon. Royal Society,
London, UK
Lamarck, J.-B.d.M, Chevalier de (1809). Philosophies zoologique, our exposition des
Considerations relatives I”histoire naturelle des animaux. Paris, Libraire.
Loewy, A. and Siekevitz, P. (1963). Cell Structure and Function. Holt, Reinhart and
Winston, New York.
Schwann, T. (1839). Mikroskopische Untersuchungen über die Uebereinstimmung in der
Struktur and dem Wachsthum der Thiere and Pflanzen. Verlag der Sander’schen
Buchbehandlung (G.E. Reimer), Berlin
B
Classification of amino acids based on R group
Based on the properties of the “R” group in each amino acid, the amino acids can be classified into five
groups:
a.Non-polar amino acids
b.Polar amino acids
c.Positively charged amino acids
d.Negatively charged amino acids
e.Aromatic amino acids
Polar amino acids
Polar amino acids have hydrophilic “R” groups, which means they prefer to interact with watery
solutions.
Non-polar amino acids
Non-polar amino acids (hydrophobic) are the polar amino acids that avoid contact with liquid. These
interactions are crucial for protein folding and give proteins their three-dimensional structure. The non-
polar amino acids are hydrophobic, whereas the rest of the amino acids are hydrophilic.
Non-polar amino acids
Ala: Alanine
Gly: Glycine
Ile: Isoleucine
Leu: Leucine
Met: Methionine
Trp: Tryptophan
Phe: Phenylalanine
Pro: Proline
Val: Valine
Polar amino acids
Cys: Cysteine
Ser: Serine
Thr: Threonine
Tyr: Tyrosine
Asn: Asparagine
Considerations relatives I”histoire naturelle des animaux. Paris, Libraire.
Loewy, A. and Siekevitz, P. (1963). Cell Structure and Function. Holt, Reinhart and
Winston, New York.
Schwann, T. (1839). Mikroskopische Untersuchungen über die Uebereinstimmung in der
Struktur and dem Wachsthum der Thiere and Pflanzen. Verlag der Sander’schen
Buchbehandlung (G.E. Reimer), Berlin
B
Classification of amino acids based on R group
Based on the properties of the “R” group in each amino acid, the amino acids can be classified into five
groups:
a.Non-polar amino acids
b.Polar amino acids
c.Positively charged amino acids
d.Negatively charged amino acids
e.Aromatic amino acids
Polar amino acids
Polar amino acids have hydrophilic “R” groups, which means they prefer to interact with watery
solutions.
Non-polar amino acids
Non-polar amino acids (hydrophobic) are the polar amino acids that avoid contact with liquid. These
interactions are crucial for protein folding and give proteins their three-dimensional structure. The non-
polar amino acids are hydrophobic, whereas the rest of the amino acids are hydrophilic.
Non-polar amino acids
Ala: Alanine
Gly: Glycine
Ile: Isoleucine
Leu: Leucine
Met: Methionine
Trp: Tryptophan
Phe: Phenylalanine
Pro: Proline
Val: Valine
Polar amino acids
Cys: Cysteine
Ser: Serine
Thr: Threonine
Tyr: Tyrosine
Asn: Asparagine
Gln: Glutamine
Polar basic amino acids (positively charged)
His: Histidine
Lys: Lysine
Arg: Arginine
Polar acidic amino acids (negatively charged)
Asp: Aspartate
Glu: Glutamate
Aromatic amino acids
Aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan, are nonpolar because of their
aromatic side chains (hydrophobic).
Classification system for amino acids based on their metabolic fate:
Glucogenic amino acids: These are precursors of gluconeogenesis, which results in glucose
production. Alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, methionine, proline, serine and valine are some of the amino acids found in
the human body.
Ketogenic amino acids: Ketone bodies are formed when these amino acids are broken down.
Leucine and Lysine are two amino acids.
Both glucogenic and ketogenic amino acids: Amino acids that are both glucogenic and ketogenic
break down to form precursors for both ketone bodies and glucose. Isoleucine, tryptophan,
phenylalanine and tyrosine are all essential amino acids.
Conclusion
Amino acids are a class of neutral substances that are separated chemically, primarily due to their
ampholytic characteristics, and biochemically, primarily due to their role as protein components, from
other natural compounds. An amino acid is a carboxylic acid with a specific stereochemistry and an
aliphatic primary amino group in the location of the carboxyl group. Proteins are biosynthesised from 20
amino acids in a genetically controlled system. As a result, amino acids are the fundamental building
blocks of proteins.
References
Binder HJ, Mansbach CM. Nutrient digestion and absorption. In: Boron WF, Boulpaep EL, eds. Medical
Physiology. 3rd ed. Philadelphia, PA: Elsevier; 2017:chap 45.
Dietzen DJ, Willrich MAV. Amino acids, peptides, and proteins. In: Rifai N, Chiu RWK, Young I, Burnham
Carey-Ann D, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. St Louis, MO: Elsevier;
2023:chap 31.
Polar basic amino acids (positively charged)
His: Histidine
Lys: Lysine
Arg: Arginine
Polar acidic amino acids (negatively charged)
Asp: Aspartate
Glu: Glutamate
Aromatic amino acids
Aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan, are nonpolar because of their
aromatic side chains (hydrophobic).
Classification system for amino acids based on their metabolic fate:
Glucogenic amino acids: These are precursors of gluconeogenesis, which results in glucose
production. Alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, methionine, proline, serine and valine are some of the amino acids found in
the human body.
Ketogenic amino acids: Ketone bodies are formed when these amino acids are broken down.
Leucine and Lysine are two amino acids.
Both glucogenic and ketogenic amino acids: Amino acids that are both glucogenic and ketogenic
break down to form precursors for both ketone bodies and glucose. Isoleucine, tryptophan,
phenylalanine and tyrosine are all essential amino acids.
Conclusion
Amino acids are a class of neutral substances that are separated chemically, primarily due to their
ampholytic characteristics, and biochemically, primarily due to their role as protein components, from
other natural compounds. An amino acid is a carboxylic acid with a specific stereochemistry and an
aliphatic primary amino group in the location of the carboxyl group. Proteins are biosynthesised from 20
amino acids in a genetically controlled system. As a result, amino acids are the fundamental building
blocks of proteins.
References
Binder HJ, Mansbach CM. Nutrient digestion and absorption. In: Boron WF, Boulpaep EL, eds. Medical
Physiology. 3rd ed. Philadelphia, PA: Elsevier; 2017:chap 45.
Dietzen DJ, Willrich MAV. Amino acids, peptides, and proteins. In: Rifai N, Chiu RWK, Young I, Burnham
Carey-Ann D, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. St Louis, MO: Elsevier;
2023:chap 31.
Trumbo P, Schlicker S, Yates AA, Poos M; Food and Nutrition Board of the Institute of Medicine, The
National Academies. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids,
cholesterol, protein and amino acids. J Am Diet Assoc. 2002;102(11):1621-1630. PMID:
12449285 pubmed.ncbi.nlm.nih.gov/12449285/.
QUESTION 2
Structure of Nucleotides
Nucleotides play central roles in many cellular processes, including metabolic regulation and the
storage and expression of genetic information. Thus, cells contain many types of nucleotides and
a complex and conflicting terminology for nucleotides and related compounds has been used,
although standard definitions aid understanding of this topic (IUPAC-IUB, 1970, 1983).
All nucleotides have three fundamental components.
1. Base: Also referred to as heterocycles, these nitrogen-containing ring compounds are
derivatives of purine or pyrimidine. The atoms within the purine/pyrimidine rings have a
common arrangement and they are given the same number in different nucleotides. A variety of
chemical groups can be bonded at different positions to the ring constituents. Although one
structure predominates for each base, tautomeric forms occur in minor amounts.
2. Sugar: A five-carbon sugar, usually ribose, is linked to the base. Each carbon atom is
numbered and, to allow their distinction from atoms in the base, the number is followed by a
prime mark: thus, ribose has five carbon atoms, numbered 1′ to 5′ . The sugars are locked into a
five-membered furanose ring by the bond from C1′ of the sugar to the base.
3. Phosphate ester: Phosphate groups are attached to the sugar by ester linkages, with the most
common site of esterification in natural compounds being via the hydroxyl at the C5′ position.
Typically, one, two or three phosphates are joined, producing mono-, di- and triphosphates,
respectively. (Note that the prefixes bis and tris are used in reference to molecules with two or
three phosphates attached to different hydroxyls on the sugar.)
In addition to their monomeric state, nucleotides exist in polymeric forms, called nucleic acids,
and there are two closely related types: ribonucleic acid (RNA) and deoxyribonucleic acid
(DNA). These polymeric forms have a directional polarity within their sequence, which occurs
because the 3′ -OH of one nucleotide is joined to the 5′ -phosphate of the next by a
phosphodiester linkage. Thus, one end of the molecule has a 5′ -phosphate and the other end has
a 3′ -OH. It is common practice that sequences are written starting with the nucleotide containing
the 5′ -phosphate at the left. Nucleotides are usually referred to in an abbreviated form. For
example, molecules with, respectively, one, two and three phosphates attached to adenosine are
AMP (adenosine monophosphate), ADP (adenosine diphosphate) and ATP (adenosine
triphosphate); the respective deoxy variants are dAMP, dADP and dATP. Nucleotides without
specified bases are written as NMP, NDP and NTP. Polynucleotides are referred to by a number
of different shortened nomenclatures, usually incorporating single capital letters to distinguish
the different.
Difference between DNA and RNA
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are perhaps the most important
molecules in cell biology, responsible for the storage and reading of genetic information that
National Academies. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids,
cholesterol, protein and amino acids. J Am Diet Assoc. 2002;102(11):1621-1630. PMID:
12449285 pubmed.ncbi.nlm.nih.gov/12449285/.
QUESTION 2
Structure of Nucleotides
Nucleotides play central roles in many cellular processes, including metabolic regulation and the
storage and expression of genetic information. Thus, cells contain many types of nucleotides and
a complex and conflicting terminology for nucleotides and related compounds has been used,
although standard definitions aid understanding of this topic (IUPAC-IUB, 1970, 1983).
All nucleotides have three fundamental components.
1. Base: Also referred to as heterocycles, these nitrogen-containing ring compounds are
derivatives of purine or pyrimidine. The atoms within the purine/pyrimidine rings have a
common arrangement and they are given the same number in different nucleotides. A variety of
chemical groups can be bonded at different positions to the ring constituents. Although one
structure predominates for each base, tautomeric forms occur in minor amounts.
2. Sugar: A five-carbon sugar, usually ribose, is linked to the base. Each carbon atom is
numbered and, to allow their distinction from atoms in the base, the number is followed by a
prime mark: thus, ribose has five carbon atoms, numbered 1′ to 5′ . The sugars are locked into a
five-membered furanose ring by the bond from C1′ of the sugar to the base.
3. Phosphate ester: Phosphate groups are attached to the sugar by ester linkages, with the most
common site of esterification in natural compounds being via the hydroxyl at the C5′ position.
Typically, one, two or three phosphates are joined, producing mono-, di- and triphosphates,
respectively. (Note that the prefixes bis and tris are used in reference to molecules with two or
three phosphates attached to different hydroxyls on the sugar.)
In addition to their monomeric state, nucleotides exist in polymeric forms, called nucleic acids,
and there are two closely related types: ribonucleic acid (RNA) and deoxyribonucleic acid
(DNA). These polymeric forms have a directional polarity within their sequence, which occurs
because the 3′ -OH of one nucleotide is joined to the 5′ -phosphate of the next by a
phosphodiester linkage. Thus, one end of the molecule has a 5′ -phosphate and the other end has
a 3′ -OH. It is common practice that sequences are written starting with the nucleotide containing
the 5′ -phosphate at the left. Nucleotides are usually referred to in an abbreviated form. For
example, molecules with, respectively, one, two and three phosphates attached to adenosine are
AMP (adenosine monophosphate), ADP (adenosine diphosphate) and ATP (adenosine
triphosphate); the respective deoxy variants are dAMP, dADP and dATP. Nucleotides without
specified bases are written as NMP, NDP and NTP. Polynucleotides are referred to by a number
of different shortened nomenclatures, usually incorporating single capital letters to distinguish
the different.
Difference between DNA and RNA
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are perhaps the most important
molecules in cell biology, responsible for the storage and reading of genetic information that
underpins all life. They are both linear polymers, consisting of sugars, phosphates and bases, but
there are some key differences which separate the two
1
. These distinctions enable the two
molecules to work together and fulfil their essential roles.
Structure
While the ubiquity of Francis Crick and James Watson’s double helix means that the two-
stranded structure of DNA structure is common knowledge, RNA’s single-stranded format is not
as well known.
RNA can form into double-stranded structures, such as during translation, when mRNA and
tRNA molecules pair. DNA polymers are also much longer than RNA polymers; the 2.3m long
human genome consists of 46 chromosomes, each of which is a single, long DNA molecule.
RNA molecules, by comparison, are much shorter
Location
Eukaryotic cells, including all animal and plant cells, house the great majority of their DNA in
the nucleus, where it exists in a tightly compressed form, called a chromosome
4
. This squeezed
format means the DNA can be easily stored and transferred. In addition to nuclear DNA, some
DNA is present in energy-producing mitochondria, small organelles found free-floating in the
cytoplasm, the area of the cell outside the nucleus.
The three types of RNA are found in different locations. mRNA is made in the nucleus, with
each mRNA fragment copied from its relative piece of DNA, before leaving the nucleus and
entering the cytoplasm. The fragments are then shuttled around the cell as needed, moved along
by the cell’s internal transport system, the cytoskeleton. tRNA, like mRNA, is a free-roaming
molecule that moves around the cytoplasm. If it receives the correct signal from the ribosome, it
will then hunt down amino acid subunits in the cytoplasm and bring them to the ribosome to be
built into proteins
5
. rRNA, as previously mentioned, is found as part of ribosomes. Ribosomes
are formed in an area of the nucleus called the nucleolus, before being exported to the cytoplasm,
where some ribosomes float freely. Other cytoplasmic ribosomes are bound to the endoplasmic
reticulum, a membranous structure that helps process proteins and export them from the cell.
Base
The nitrogen bases in DNA are the basic units of genetic code, and their correct ordering and
pairing is essential to biological function. The four bases that make up this code are adenine (A),
thymine (T), guanine (G) and cytosine (C). Bases pair off together in a double helix structure,
these pairs being A and T, and C and G. RNA doesn’t contain thymine bases, replacing them
with uracil bases (U), which pair to adenine
B
Many discoveries led to the uncovering of the double-stranded structure of DNA, proposed by J
Watson and F Crick in 1953, and more followed to build the foundations for molecular medicine.
DNA comprises two polynucleotide strands twisted around each other in the form of a double
helix. Double-stranded DNA refers to the major form of genetic material in most organisms,
where two strands of DNA are joined together. It is characterized by having a single replication
origin in prokaryotic chromosomes and multiple replication origins dispersed throughout
eukaryotic chromosomes. In biological terms, the double-stranded DNA structure is essential for
replication to ensure that each dividing cell receives an identical copy of the DNA
there are some key differences which separate the two
1
. These distinctions enable the two
molecules to work together and fulfil their essential roles.
Structure
While the ubiquity of Francis Crick and James Watson’s double helix means that the two-
stranded structure of DNA structure is common knowledge, RNA’s single-stranded format is not
as well known.
RNA can form into double-stranded structures, such as during translation, when mRNA and
tRNA molecules pair. DNA polymers are also much longer than RNA polymers; the 2.3m long
human genome consists of 46 chromosomes, each of which is a single, long DNA molecule.
RNA molecules, by comparison, are much shorter
Location
Eukaryotic cells, including all animal and plant cells, house the great majority of their DNA in
the nucleus, where it exists in a tightly compressed form, called a chromosome
4
. This squeezed
format means the DNA can be easily stored and transferred. In addition to nuclear DNA, some
DNA is present in energy-producing mitochondria, small organelles found free-floating in the
cytoplasm, the area of the cell outside the nucleus.
The three types of RNA are found in different locations. mRNA is made in the nucleus, with
each mRNA fragment copied from its relative piece of DNA, before leaving the nucleus and
entering the cytoplasm. The fragments are then shuttled around the cell as needed, moved along
by the cell’s internal transport system, the cytoskeleton. tRNA, like mRNA, is a free-roaming
molecule that moves around the cytoplasm. If it receives the correct signal from the ribosome, it
will then hunt down amino acid subunits in the cytoplasm and bring them to the ribosome to be
built into proteins
5
. rRNA, as previously mentioned, is found as part of ribosomes. Ribosomes
are formed in an area of the nucleus called the nucleolus, before being exported to the cytoplasm,
where some ribosomes float freely. Other cytoplasmic ribosomes are bound to the endoplasmic
reticulum, a membranous structure that helps process proteins and export them from the cell.
Base
The nitrogen bases in DNA are the basic units of genetic code, and their correct ordering and
pairing is essential to biological function. The four bases that make up this code are adenine (A),
thymine (T), guanine (G) and cytosine (C). Bases pair off together in a double helix structure,
these pairs being A and T, and C and G. RNA doesn’t contain thymine bases, replacing them
with uracil bases (U), which pair to adenine
B
Many discoveries led to the uncovering of the double-stranded structure of DNA, proposed by J
Watson and F Crick in 1953, and more followed to build the foundations for molecular medicine.
DNA comprises two polynucleotide strands twisted around each other in the form of a double
helix. Double-stranded DNA refers to the major form of genetic material in most organisms,
where two strands of DNA are joined together. It is characterized by having a single replication
origin in prokaryotic chromosomes and multiple replication origins dispersed throughout
eukaryotic chromosomes. In biological terms, the double-stranded DNA structure is essential for
replication to ensure that each dividing cell receives an identical copy of the DNA
C
Protein synthesis
Protein synthesis is an energy intensive process requiring about 10 translation factors in addition
to aminoacyl-tRNAs and the ribosome. The process of protein synthesis can be divided into four
stages: (1) initiation, (2) elongation, (3) termination, and (4) recycling. During the initiation stage
of protein synthesis, the 30S ribosomal subunit binds the mRNA and the initiator tRNA (formyl-
methionine-tRNA) with the help of initiation factors 1, 2, and 3 (IF1, IF2, and IF3) (reviewed
in Boelens and Gualerzi, 2002). The initiator tRNA binds to the P site in the 30S subunit and
interacts with the start codon in the mRNA. Correct base pair formation between
the anticodon of the initiator tRNA and the start codon promotes the association of the 50S
subunit to the 30S subunit and the release of IF1, IF2, and IF3 from the ribosome. The ribosome
then begins the elongation cycle of protein synthesis by binding elongation factor
Tu•GTP•aminoacyl tRNA (EF-Tu ternary complex) to the ribosomal A site. Proper Watson–
Crick base pair formation between the anticodon of the tRNA and the mRNA codon in the A site
triggers the accommodation of the tRNA into the 50S subunit and the release of EF-Tu•GDP
from the ribosome. The peptidyl transferase center in the 50S subunit then catalyzes peptide
bond formation resulting in the extension of the nascent peptide by one amino acid. Next, the
deacylated tRNA in the P site and the peptidyl-tRNA in the A site are translocated to the E and P
sites, respectively, by the activity of elongation factor G (EF-G). This process also brings the
next mRNA codon into the A site for interaction with EF-Tu ternary complexes. This cycle
continues until a stop codon is placed in the A site. The stop codon in the A site is recognized not
by an EF-Tu ternary complex but by release factors 1 or 2 (RF1 recognizes UAA and UAG
where as RF2 recognizes UAA and UGA). RF1 and RF2 promote the release of the newly
synthesized protein from the peptidyl-tRNA in the P site. RF1 and RF2 are released from the
ribosome by the activity of RF3•GTP, which in turn hydrolyze GTP to dissociate from the
ribosome. The ribosome is now left with a deacylated tRNA in the P site and the mRNA. This
ribosomal complex binds RRF and EF-G, which separates the ribosome into the 50S and 30S
subunits and the mRNA and tRNA are released from the 30S subunit by the proofreading
activity of IF3. The vacant 50S and 30S subunits are free to start the whole process of protein
synthesis again on another mRNA.
Question 3
Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by
molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of the
enzymes to cut DNA at precise locations enabled researchers to isolate gene-containing
fragments and recombine them with other molecules of DNA—i.e., to clone genes. The names of
restriction enzymes are derived from the genus, species, and strain designations of the bacteria
that produce them; for example, the enzyme EcoRI is produced by Escherichia coli strain RY13.
It is thought that restriction enzymes originated from a common ancestral protein and evolved to
recognize specific sequences through processes such as genetic recombination and gene
amplification.
Restriction enzyme, a protein produced by bacteria that cleaves DNA at specific sites along
the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating
Protein synthesis
Protein synthesis is an energy intensive process requiring about 10 translation factors in addition
to aminoacyl-tRNAs and the ribosome. The process of protein synthesis can be divided into four
stages: (1) initiation, (2) elongation, (3) termination, and (4) recycling. During the initiation stage
of protein synthesis, the 30S ribosomal subunit binds the mRNA and the initiator tRNA (formyl-
methionine-tRNA) with the help of initiation factors 1, 2, and 3 (IF1, IF2, and IF3) (reviewed
in Boelens and Gualerzi, 2002). The initiator tRNA binds to the P site in the 30S subunit and
interacts with the start codon in the mRNA. Correct base pair formation between
the anticodon of the initiator tRNA and the start codon promotes the association of the 50S
subunit to the 30S subunit and the release of IF1, IF2, and IF3 from the ribosome. The ribosome
then begins the elongation cycle of protein synthesis by binding elongation factor
Tu•GTP•aminoacyl tRNA (EF-Tu ternary complex) to the ribosomal A site. Proper Watson–
Crick base pair formation between the anticodon of the tRNA and the mRNA codon in the A site
triggers the accommodation of the tRNA into the 50S subunit and the release of EF-Tu•GDP
from the ribosome. The peptidyl transferase center in the 50S subunit then catalyzes peptide
bond formation resulting in the extension of the nascent peptide by one amino acid. Next, the
deacylated tRNA in the P site and the peptidyl-tRNA in the A site are translocated to the E and P
sites, respectively, by the activity of elongation factor G (EF-G). This process also brings the
next mRNA codon into the A site for interaction with EF-Tu ternary complexes. This cycle
continues until a stop codon is placed in the A site. The stop codon in the A site is recognized not
by an EF-Tu ternary complex but by release factors 1 or 2 (RF1 recognizes UAA and UAG
where as RF2 recognizes UAA and UGA). RF1 and RF2 promote the release of the newly
synthesized protein from the peptidyl-tRNA in the P site. RF1 and RF2 are released from the
ribosome by the activity of RF3•GTP, which in turn hydrolyze GTP to dissociate from the
ribosome. The ribosome is now left with a deacylated tRNA in the P site and the mRNA. This
ribosomal complex binds RRF and EF-G, which separates the ribosome into the 50S and 30S
subunits and the mRNA and tRNA are released from the 30S subunit by the proofreading
activity of IF3. The vacant 50S and 30S subunits are free to start the whole process of protein
synthesis again on another mRNA.
Question 3
Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by
molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of the
enzymes to cut DNA at precise locations enabled researchers to isolate gene-containing
fragments and recombine them with other molecules of DNA—i.e., to clone genes. The names of
restriction enzymes are derived from the genus, species, and strain designations of the bacteria
that produce them; for example, the enzyme EcoRI is produced by Escherichia coli strain RY13.
It is thought that restriction enzymes originated from a common ancestral protein and evolved to
recognize specific sequences through processes such as genetic recombination and gene
amplification.
Restriction enzyme, a protein produced by bacteria that cleaves DNA at specific sites along
the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating
infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the
laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason
they are indispensible tools of recombinant DNA technology (genetic engineering).
A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages,
or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it
might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it
into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the
number of strains of bacteriophage that can infect a bacterium. Each restriction enzyme
recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the
linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions
are called recognition sequences, or recognition sites, and are randomly distributed throughout
the DNA. Different bacterial species make restriction enzymes that recognize different
nucleotide sequences.
When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by
catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the
bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in this
manner by disguising their recognition sequences. Enzymes called methylases add methyl
groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus
modified and protected from the endonuclease. The restriction enzyme and its corresponding
methylase constitute the restriction-modification system of a bacterial species.
Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV,
which differ primarily in structure, cleavage site, specificity, and cofactors. Types I and III
enzymes are similar in that both restriction and methylase activities are carried out by one
large enzyme complex, in contrast to the type II system, in which the restriction enzyme is
independent of its methylase. Type II restriction enzymes also differ from types I and III in that
they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly,
sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction
enzymes have been identified from a variety of bacterial species. These enzymes recognize a few
hundred distinct sequences, generally four to eight bases in length. Type IV restriction enzymes
cleave only methylated DNA and show weak sequence specificity.
Restriction enzymes are one of the easiest approaches to detect modified DNA at specific
genomic sites. Cleavage of DNA by a restriction enzyme may be blocked or impaired when a
particular base in the recognition site is modified. For example, MspI and HpaII recognize the
same sequence (CCGG); however, they are sensitive to different modification status: when the
external C in the sequence CCGG is methylated, MspI and HpaII cannot cleave. Unlike HpaII,
MspI can cleave the sequence when the internal C residue is methylated (Bird & Southern,
1978). Another enzyme, Pvurts1I, only cleave the sequence
hm
CN11–12/N9–10G, which
contains 5hmC (Asgar Abbas, Monika, Honorata, & Matthias, 2014; Evelina & Giedrius, 2014;
Sun et al., 2015). The combination of DpnI and DpnII is use to detect m6dA; both recognize the
consensus sequence GATC, but only DpnI will cleave at this site if the adenine is methylated (Fu
et al., 2015; Greer et al., 2015; Heyn & Esteller, 2015; Ratel, Ravanat, Berger, & Wion, 2006).
Thus, using different restriction enzymes, we could detect DNA modification beyond
familiar cytosine, including 5mC and 5hmC. In addition, this approach is cost-effective and fast;
laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason
they are indispensible tools of recombinant DNA technology (genetic engineering).
A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages,
or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it
might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it
into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the
number of strains of bacteriophage that can infect a bacterium. Each restriction enzyme
recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the
linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions
are called recognition sequences, or recognition sites, and are randomly distributed throughout
the DNA. Different bacterial species make restriction enzymes that recognize different
nucleotide sequences.
When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by
catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the
bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in this
manner by disguising their recognition sequences. Enzymes called methylases add methyl
groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus
modified and protected from the endonuclease. The restriction enzyme and its corresponding
methylase constitute the restriction-modification system of a bacterial species.
Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV,
which differ primarily in structure, cleavage site, specificity, and cofactors. Types I and III
enzymes are similar in that both restriction and methylase activities are carried out by one
large enzyme complex, in contrast to the type II system, in which the restriction enzyme is
independent of its methylase. Type II restriction enzymes also differ from types I and III in that
they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly,
sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction
enzymes have been identified from a variety of bacterial species. These enzymes recognize a few
hundred distinct sequences, generally four to eight bases in length. Type IV restriction enzymes
cleave only methylated DNA and show weak sequence specificity.
Restriction enzymes are one of the easiest approaches to detect modified DNA at specific
genomic sites. Cleavage of DNA by a restriction enzyme may be blocked or impaired when a
particular base in the recognition site is modified. For example, MspI and HpaII recognize the
same sequence (CCGG); however, they are sensitive to different modification status: when the
external C in the sequence CCGG is methylated, MspI and HpaII cannot cleave. Unlike HpaII,
MspI can cleave the sequence when the internal C residue is methylated (Bird & Southern,
1978). Another enzyme, Pvurts1I, only cleave the sequence
hm
CN11–12/N9–10G, which
contains 5hmC (Asgar Abbas, Monika, Honorata, & Matthias, 2014; Evelina & Giedrius, 2014;
Sun et al., 2015). The combination of DpnI and DpnII is use to detect m6dA; both recognize the
consensus sequence GATC, but only DpnI will cleave at this site if the adenine is methylated (Fu
et al., 2015; Greer et al., 2015; Heyn & Esteller, 2015; Ratel, Ravanat, Berger, & Wion, 2006).
Thus, using different restriction enzymes, we could detect DNA modification beyond
familiar cytosine, including 5mC and 5hmC. In addition, this approach is cost-effective and fast;
however, it is still limited by the number and distribution of restriction sites in the genome. Dai
et al. (2002) found that a maximum of 4100 sites can be accessed by the restriction enzymes
known to be DNA modification sensitive, and specific sites of interest that are not located at
restriction sites cannot be investigated using this method.
B
Gene Isolation
Genes for large hormones, proteins, and enzymes could be made by chemical DNA
synthesis (synthesis rate about one nucleotide per day per person). However, in most cases
probably it will be preferable to isolate the natural DNA sequence by cloning a reverse transcript
of the appropriate mRNA. This approach to gene isolation works well if a method is available for
detecting the desired gene sequence among the “shotgun” library of bacterial or viral clones.
Detection of the desired clone usually is difficult except for the most abundant protein (and thus
mRNA) species. With present methodology, it is very difficult to isolate a specific gene whose
mRNA transcript is not present as more than 1% of the total mRNA.
Synthetic DNA, however, may allow the isolation of the natural gene for any protein for which at
least partial amino acid sequence is known. Using the genetic code, a short (10–20
nucleotide) synthetic DNA fragment can be made that is complementary to the mRNA of the
desired gene. Degeneracy of the genetic code will require that a mixture of synthetic probes be
made for most proteins, but this seems feasible. Synthetic probes have been used to identify
the cytochrome c gene of yeast (Montgomery et al., 1978). Noyes et al. (1979) have used a
synthetic oligonucleotide to specifically prime gastrin cDNA synthesis.
A major route to gene isolation relies on fine-scale genetic mapping, and this approach is
particularly suited to the situation where the phenotype generated by the target gene is defined,
but the biochemical mechanism of its action is not understood. The strategy relies on being able
to identify genetic markers very closely linked to and, if possible, flanking the target gene, and
this close linkage can only be achieved by mapping in large populations. These populations are
typically an order of magnitude larger than a normal mapping population, but the number of loci
included in these fine-scale maps is necessarily very small, since only those linked to the target
are included. Commonly, a bulk segregant strategy is applied to identify critical markers; for this
purpose, DNA from individuals with each of the two alternative alleles is pooled, and the two
pools are then fingerprinted. A marker(s) which differentiates one pool from the other is likely to
be linked to the target, since the “background” of the two pools is identical. Closely linked
flanking markers represent the starting points of a chromosome walk, which is effected by
assembling a contig of large insert clones spanning the region containing the target.
Gene cloning
Any DNA fragment that contains a gene of interest can be cloned. In cell biology, the term DNA
cloning is used in two senses. In one sense it literally refers to the act of making many identical
copies of a DNA molecule—the amplification of a particular DNA sequence. However, the term
is also used to describe the isolation of a particular stretch of DNA (often a particular gene) from
the rest of a cell's DNA, because this isolation is greatly facilitated by making many identical
copies of the DNA of interest.
et al. (2002) found that a maximum of 4100 sites can be accessed by the restriction enzymes
known to be DNA modification sensitive, and specific sites of interest that are not located at
restriction sites cannot be investigated using this method.
B
Gene Isolation
Genes for large hormones, proteins, and enzymes could be made by chemical DNA
synthesis (synthesis rate about one nucleotide per day per person). However, in most cases
probably it will be preferable to isolate the natural DNA sequence by cloning a reverse transcript
of the appropriate mRNA. This approach to gene isolation works well if a method is available for
detecting the desired gene sequence among the “shotgun” library of bacterial or viral clones.
Detection of the desired clone usually is difficult except for the most abundant protein (and thus
mRNA) species. With present methodology, it is very difficult to isolate a specific gene whose
mRNA transcript is not present as more than 1% of the total mRNA.
Synthetic DNA, however, may allow the isolation of the natural gene for any protein for which at
least partial amino acid sequence is known. Using the genetic code, a short (10–20
nucleotide) synthetic DNA fragment can be made that is complementary to the mRNA of the
desired gene. Degeneracy of the genetic code will require that a mixture of synthetic probes be
made for most proteins, but this seems feasible. Synthetic probes have been used to identify
the cytochrome c gene of yeast (Montgomery et al., 1978). Noyes et al. (1979) have used a
synthetic oligonucleotide to specifically prime gastrin cDNA synthesis.
A major route to gene isolation relies on fine-scale genetic mapping, and this approach is
particularly suited to the situation where the phenotype generated by the target gene is defined,
but the biochemical mechanism of its action is not understood. The strategy relies on being able
to identify genetic markers very closely linked to and, if possible, flanking the target gene, and
this close linkage can only be achieved by mapping in large populations. These populations are
typically an order of magnitude larger than a normal mapping population, but the number of loci
included in these fine-scale maps is necessarily very small, since only those linked to the target
are included. Commonly, a bulk segregant strategy is applied to identify critical markers; for this
purpose, DNA from individuals with each of the two alternative alleles is pooled, and the two
pools are then fingerprinted. A marker(s) which differentiates one pool from the other is likely to
be linked to the target, since the “background” of the two pools is identical. Closely linked
flanking markers represent the starting points of a chromosome walk, which is effected by
assembling a contig of large insert clones spanning the region containing the target.
Gene cloning
Any DNA fragment that contains a gene of interest can be cloned. In cell biology, the term DNA
cloning is used in two senses. In one sense it literally refers to the act of making many identical
copies of a DNA molecule—the amplification of a particular DNA sequence. However, the term
is also used to describe the isolation of a particular stretch of DNA (often a particular gene) from
the rest of a cell's DNA, because this isolation is greatly facilitated by making many identical
copies of the DNA of interest.
DNA cloning in its most general sense can be accomplished in several ways. The simplest
involves inserting a particular fragment of DNA into the purified DNA genome of a self-
replicating genetic element—generally a virus or a plasmid. A DNA fragment containing a
human gene, for example, can be joined in a test tube to the chromosome of a bacterial virus, and
the new recombinant DNA molecule can then be introduced into a bacterial cell. Starting with
only one such recombinant DNA molecule that infects a single cell, the normal replication
mechanisms of the virus can produce more than 10
12
identical virus DNA molecules in less than
a day, thereby amplifying the amount of the inserted human DNA fragment by the same factor.
A virus or plasmid used in this way is known as a cloning vector, and the DNA propagated by
insertion into it is said to have been cloned.
To isolate a specific gene, one often begins by constructing a DNA library—a comprehensive
collection of cloned DNA fragments from a cell, tissue, or organism. This library includes (one
hopes) at least one fragment that contains the gene of interest. Libraries can be constructed with
either a virus or a plasmid vector and are generally housed in a population of bacterial cells. The
principles underlying the methods used for cloning genes are the same for either type of cloning
vector, although the details may differ. Today most cloning is performed with plasmid vectors.
The plasmid vectors most widely used for gene cloning are small circular molecules of double-
stranded DNA derived from larger plasmids that occur naturally in bacterial cells. They generally
account for only a minor fraction of the total host bacterial cell DNA, but they can easily be
separated owing to their small size from chromosomal DNA molecules, which are large and
precipitate as a pellet upon centrifugation. For use as cloning vectors, the purified plasmid DNA
circles are first cut with a restriction nuclease to create linear DNA molecules. The cellular DNA
to be used in constructing the library is cut with the same restriction nuclease, and the resulting
restriction fragments (including those containing the gene to be cloned) are then added to the cut
plasmids and annealed via their cohesive ends to form recombinant DNA circles. These
recombinant molecules containing foreign DNA inserts are then covalently sealed with the
enzyme DNA ligase
involves inserting a particular fragment of DNA into the purified DNA genome of a self-
replicating genetic element—generally a virus or a plasmid. A DNA fragment containing a
human gene, for example, can be joined in a test tube to the chromosome of a bacterial virus, and
the new recombinant DNA molecule can then be introduced into a bacterial cell. Starting with
only one such recombinant DNA molecule that infects a single cell, the normal replication
mechanisms of the virus can produce more than 10
12
identical virus DNA molecules in less than
a day, thereby amplifying the amount of the inserted human DNA fragment by the same factor.
A virus or plasmid used in this way is known as a cloning vector, and the DNA propagated by
insertion into it is said to have been cloned.
To isolate a specific gene, one often begins by constructing a DNA library—a comprehensive
collection of cloned DNA fragments from a cell, tissue, or organism. This library includes (one
hopes) at least one fragment that contains the gene of interest. Libraries can be constructed with
either a virus or a plasmid vector and are generally housed in a population of bacterial cells. The
principles underlying the methods used for cloning genes are the same for either type of cloning
vector, although the details may differ. Today most cloning is performed with plasmid vectors.
The plasmid vectors most widely used for gene cloning are small circular molecules of double-
stranded DNA derived from larger plasmids that occur naturally in bacterial cells. They generally
account for only a minor fraction of the total host bacterial cell DNA, but they can easily be
separated owing to their small size from chromosomal DNA molecules, which are large and
precipitate as a pellet upon centrifugation. For use as cloning vectors, the purified plasmid DNA
circles are first cut with a restriction nuclease to create linear DNA molecules. The cellular DNA
to be used in constructing the library is cut with the same restriction nuclease, and the resulting
restriction fragments (including those containing the gene to be cloned) are then added to the cut
plasmids and annealed via their cohesive ends to form recombinant DNA circles. These
recombinant molecules containing foreign DNA inserts are then covalently sealed with the
enzyme DNA ligase
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