III Pharm D (2011-2012), Pharmacology-II (T), PESCP, Bangalore-50, KPS Gowda

Asst.Prof.

6. The dynamic cell: The structures and functions of the components of the cell

a) Cell and macromolecules: Cellular classification, subcellular organelles,macromolecules,

large macromolecular assemblies b) Chromosome structure: Pro and eukaryotic chromosome

structures, chromatin structure, genome complexity, the flow of genetic information. c) DNA

replication: General, bacterial and eukaryotic DNA replication. d) The cell cycle: Restriction

point, cell cycle regulators and modifiers. e) Cell signaling: Communication between cells and

their environment, ionchannels, signal transduction pathways (MAP kinase, P38 kinase, JNK,

Ras and PI3-kinase pathways, biosensors.

a)Cell and macromolecules: Cellular classification, subcellular organelles, macromolecules,

large macromolecular assemblies- The cell comes from the Latin cellula, meaning “a small

room”. The cell is the structural and functional unit of all known living organisms. The cell was

discovered by Robert Hooke in 1665. Human contains about 10 trillion cells. Most plant and

animal cells are between 1 and 100µm and therefore are visible only under the microscope.

Types of cell: There are two types of cell. Prokaryotic and eukaryotic.

Prokaryotic cells: Prokaryotic word derived from Greek meaning- before nuclei. Examples-

cells in the bacteria and cyano bacteria (blue green algae).These cells have few internal

structures. They do not have membrane bound nucleus. The bacterial cells are very small (about

1-2 µm diameter and 10 µm long). These cells have 3 shapes- rod, spherical and spiral. The cell

division is by binary fission.

The structural components of prokaryotic cells: The nuclear material of prokaryotic cell

consists of a single chromosome. The flagella and pili are projected from the cell surface. These

consist of proteins. They facilitate the movement and communication between the cells. The

cells enclosed by cell envelop. This consists of plasma membrane and cell wall. Some bacteria

also have another layer- capsule. This envelop gives the rigidity to the cell. Inside the cell is the

cytoplasmic region that contains the cell genome (DNA), ribosomes and various cell inclusions.

The DNA is condensed to a nucleiod. There are circular structures called plasmids, which carry

extrachromosomal DNA.

2. Eukaryotic cells: Eukaryotic word is derived from Greek- eu means good and karyon means

nut or kernel. These cells contain complex structures enclosed within the membranes. The

membrane bound nucleus is present in these cells. Most of these cells also contain other

membrane bound organelles such as mitochondria, chloroplast and the Golgi apparatus. All

species of large complex organisms are eukaryotes, including animals, plants and fungi.

Organelles of the eukaryotic cells: 1.Lysosomes: These are cellular organelles that contain

acid hydrolase enzymes to break down waste materials and cellular debris. They are found in

animal cells. The membrane around a lysosome allows the digestive enzymes to work at the

acidic pH. The lysosomes are formed from the Golgi apparatus. The aged cell organelles are

degraded in a lysosome is called autophagy. They are also called as suicide-bags or suicide-sacs

due to their autolysis. The size of the lysosomes varies from 0.1 to 1.2µm. The pH of the interior

of the lysosomes is 4.8 and it is acidic compared to the slightly alkaline cytosol (pH 7.2). The

lysosome maintains this pH differential by pumping protons (H+ ions) from the cytosol across

the membrane via proton pumps and chloride ion channels. The lysosomal membrane protects

the cytosol, and rest of the cell, from the degradative enzymes of the lysosome. If lysosomal acid

hydrolases of lysosomes leaked into cytosol , these enzymes fails to produce their effects in the

alkaline environment. Some of the examples of digestion by the acid hydrolase enzymes.-

Nuclease degrade RNA and DNA into their mononucleotides building blocks. Protease degrades

proteins to peptides. Phosphatases remove phosphate groups from mononucleotides,

phospholipids and other compounds.

Tay-Sachs disease is caused by the deficiency of lysosomal enzyme that digests gangliosides (a

glycolipid). This results in the accumulation of these glycolipids in the neurons. It is inherited

disease; the affected children commonly become demented and blind by age 2 and die before

their 3rd

birthday.

Autophagy- It is a catabolic process involving degradation of a cell’s own components through

lysosomal enzymes. It involves the formation of a membrane around a targeted region of the cell,

separating the contents from the rest of the cytoplasma. The resultant vesicle fuses with a lysome

and subsequently degrades the contents.

Autolysis- It is the spontaneous disintegration of cells or tissues by lysosomal enzymes, as

occurs after death and in some pathological conditions.

Peroxisomes: These are roughly spherical organelles. All animal cells (except RBC) contain

peroxisomes. Its diameter is 0.2 to 1µm.Peroxisomes contain several oxidases-enzymes that use

molecular oxygen to oxidize organic substances, in the process forming H2O2. Peroxisome also

contains catalase, which degrades hydrogen peroxide to yield water and oxygen.

2H2O2 -------- catalase2H2O +O2

In X-linked adrenoleukodystrophy (ADL), the peroxisomal oxidation of very long chain fatty

acids is defective. This damages the white matter of the brain and impairs the adrenal glands.

3.Endoplasmic reticulum: It is an extensive network of closed, flattened membrane-bounded

sacs called cisternae. In smooth ER the synthesis of fatty acids and phospholipids takes place.

The hepatocytes have abudant smooth ER. Enzymes in the smooth ER of the liver also modify or

detoxify hydrophobic chemicals such as pesticides and carcinogens into more water soluble,

conjugated products that can be excreted from the body. The smooth ER is one of the sites of

drug metabolism by cytochrome-450 enzymes. High doses of such compounds result in

enlargement of smooth ER in the liver cells.

The proteins manufacturing ribosome are present on the surface of the rough ER. The ribosome

are bind with the riboporin (a glycoprotein receptor) expressed on the surface of the RER. These

receptors are not present on the smooth ER

The functions of RER – It facilitates the folding of newly synthesized proteins.Only properly

folded proteins are transported from RER to Golgi complex.RER helps for the transportation of

newly synthesized proteins. The RER helps for the insertion of proteins into the endoplasmic

reticulum membrane. It helps for glycosylation- attachment of oligosaccharides to a protein. It

helps for disulfide bond formation, as this helps for the stabilization of tertiary and quaternary

structure of the proteins.

4.The Golgi complex- It is the cell organelle of the eukayotic cells. It was identified by Italian

Physician Camello Golgi in 1898. The stack of Golgi cisternae has 3 regions- the cis, the medial,

and the trans. Transport vesicles from the RER fuse with the cis region of the Golgi complex,

where they deposit their protein contents. These proteins then progress from the cis to the medial

and then to the trans region. Within each region different enzymes modify the proteins. The

modified proteins get packed into the secretory vesicles. The functions of Golgi complex are

1.Enzymes present in the cisternae modify proteins- glycosylation,phosphorylation,etc. 2.

Transportation of lipids within the cell. 3. Formation of lysosomes. 4. Play a role in the synthesis

of the proteoglycons and carbohydrates.

5.Mitochondria (singular-mitochondrion)- Mitochondrion is derived from–mitos meaning

thread, chondrion- granule. These are the membrane-enclosed organelles found in the eukaryotic

cells. Size - 0.5 to 1 micrometer (µm) in diameter. These are described as power houses of the

cell, as they generate ATP. The two membranes that bound a mitochondrion differ in

composition and function. The outer membrane composed of about half lipids and half proteins.

This is permeable to molecules having molecular weight as high as 10,000. The inner membrane

is less permeable, is composed of 20% lipids and 80% proteins. The large number of in folding

of the inner membrane (cristae) increases the surface area. The ATPs are synthesized from fatty

acids and glucose. The complete aerobic degradation of glucose to CO2 and H2O leads to 30

molecules of ATP. In eukaryotic cells, the initial stages of glucose degradation takes place in the

cytosol, where 2 ATP molecules per glucose are generated. The terminal stages of oxidation and

synthesis of ATP are carried out by enzymes in the mitochondrial matrix and inner membrane.

28 ATP molecules per glucose molecule are generated in mitochondria.

6.Cytoskeleton: The eukaryotic cells contain 3 kinds of cytoskeletal structures, which are

microfilaments, intermediate filaments and microtubules.

a.Microfilaments: (actin filaments)- These are solid rods about 7nm in diameter. They are also

called actin filaments, because they are built from molecules called actin (a protein). A

microfilament is a twisted double chain of actin subunits. Microfilaments are present in all

eukaryotic-

cells.

b.Intermediate filaments. Its average size is 10nm. It is mainly present in the cytoplasm. But

lamin intermediate filament found in the nucleus.

c.Microtubules: Hallow tube like structures, 24nm diameter.

Each type of cytoskeletal filament is a polymer of protein subunits. Monomeric actin subunits

assemble into microfilaments, dimeric subunits composed of α and β-tubulin polymerize into

microtubules. The intermediate filaments are composed of different proteins, examples- lamins,

keratin, etc. The cytoskeleton provides shape to the cell and helps for the movement the cell

organells and cell.

7.Nucleus: It is the largest cell organelle in animal cells, is surrounded by two membrane made

up of phospholipids and proteins. The two nuclear membranes appear to fuse at nuclear pores,

through which material moves between the nucleus and the cytosol. In a growing or

differentiating cell, the nucleus is metabolically active, replicating DNA and synthesizing rRNA,

tRNA, and mRNA. Most of the ribosomal RNA is synthesized in the nucleolus. The DNA is

packaged into chromosomes. In a nucleus that is not dividing, the chromosomes are dispersed.

During cell division the individual chromosomes are visible by light microscope.

Macromolecules: Organic molecules are molecules that contain carbon and hydrogen. All living

things contain these organic molecules. Small organic molecules can combine into very large

molecules that are called macromolecules. Macromolecules are usually polymers (poly= many,

mers= parts). A polymer is a large molecule formed by the covalent bonding of many identical or

similar small building-block molecules called monomers. Usually the reaction that joins two

monomers is a dehydration synthesis. In this type of reaction, hydrogen is removed from one

monomer and a hydroxyl group is removed from other to form a water molecule.

Macromolecules such as carbohydrates, lipids, proteins, and nucleic-acids are assembled in cells

via dehydration synthesis reactions. There are four basic kinds of biological macromolecules.

They are carbohydrates, lipids, proteins and nucleic acids. The polymers are composed of

different monomers and serve different functions.

Carbon

Carbon has four electrons in its outer shell.

Hydrogen has one electron and one proton.

Carbon can bond by covalent bonds with as many as 4 other atoms.

Methane molecule.

Carbon also form double covalent or triple covalent bonds..

Carbon can form 4 covalent bonds because it has 4 electrons in its outer shell. It can form the

following number of bonds..

4 single bonds

2 double bonds

1 double bond and 2 single bonds

1 triple bond and 1 single bond

Long chains of carbon are common. The chains may be branched or form

rings.

Hydrophilic and hydrophobic:

Polar and ionic molecules have positive and negative charges and are therefore attracted to water

molecules because water molecules are also polar. They are said to be hydrophilic because they

interact with (dissolve in ) water by forming hydrogen bonds.

Non polar molecules are hydrophobic (means “water fearing”). They do not dissolve in water.

Non polar molecules are hydrophobic.

Polar and ionic molecules are hydrophilic.

Functional groups

Organic molecules may have functional groups attached. Example, COOH is a carboxyl group.

The letter R is used to indicate an organic molecule. For example, the diagram below can

represent a carboxylic group. The R can be any organic molecule.

If polar or ionizing functional groups are attached to hydrophobic molecules, the molecule may

become hydrophilic due to the functional group. Some ionizing functional groups are:- COOH,

OH, CO and NH2. Some important functional groups are shown below.

Name

Structure

Non-ionized Ionized

Hydroxyl

Carboxyl

Amino

Phosphate

Sulfhydryl

Aldehyde

Ketone

Isomers - Different molecules that are composed of the same number and kinds of atoms are

called isomers. Glucose and fructose are both C6H12O6 but the atoms are arranged differently in

each molecule.

Structural isomers differ in their overall construction as shown above for glucose and fructose.

Geometric isomers maintain the same carbon skeleton but a double bond occurs between carbon

atoms. The two molecules below are geometric isomers because the double bond cannot rotate. If

the bond between the two carbon atoms were a single bond, they would not be isomers because

atoms attached by single bonds can rotate.

Enantiomers are molecules that are mirror images of each other. The molecules shown below are

enantiomers.

Condensations- In order to bond the two molecules together, the first hydrogen of each molecule

is removed. This is necessary because carbon has a maximum of 4 bonds and hydrogen can have

only one.

In biological systems, macromolecules are formed by removing H from one atom and OH from

the other. The H and the OH combine to form water. Small molecules (monomers) are therefore

joined to build macromolecules by the removal of water. Example: The sugar (sucrose) can be

produced by a condensation reaction of glucose and

fructose.

Sucrose:

Hydrolysis

This is a type of reaction in which a macromolecule is broken down into small molecule. It is the

reverse of condensation.

Macromolecules and monomers: Many of the common biological (macromolecules) are

synthesized from simpler building blocks (monomers).

Example of a macromolecule monomer

Polysaccharide Monosaccharide (simple sugar)

Fat(a lipid) Glycerol, fatty acid

Protein Amino acid

Nucleic acid Nucleotide

Carbohydrates- The general formula for carbohydrates is (CH2O)n

Carbohydrates include sugars, glycogen, starches, and cellulose. They represent only 2-3% of

total body mass. In humans and animals, carbohydrates function mainly as a source of chemical

energy for generating ATP needed to drive metabolic reactions. Only few carbohydrates are used

for building structural units. Examples- Deoxyribose of DNA and ribose of RNA.

Monosaccharide and disaccharides are known as simple sugars. Oligosaccharides made up of 2-

10 and polysacharides made up of more than 10 monosaccharides.

Monosaccharides: Glucose (the main blood sugar), fructose (found in fruits), galactose (milk

sugar), deoxyribose (in DNA), ribose (in RNA). The names of most sugars end with the letters

ose

There are two isomers of the ring form of glucose. They differ in the location of the OH group on

the number 1 carbon atom.

The number 1 carbon atom of the linear form of glucose is attached to the oxygen on the number

5 carbon atom. Simple sugars store energy for cells. Cells also use simple sugars to construct

other kinds of organic molecules.(eg. Ribose and 2-deoxyribose).

Disaccharides- Disaccharides are composed of 2 monosaccharides joined together by a

condensation reaction.Examples: Sucrose (table sugar) is composed of glucose and fructose.

Like glucose, sucrose stores energy.

Lactose is found in milk. It is formed when glucose binds to galactose. The digestion of

carbohydrates involves hydrolysis reactions in which complex carbohydrates (polysaccharides)

are broken down to maltose (a disaccharide). Maltose is then further broken down to produce

two glucose molecules.

Polysaccharides-

Monosaccharides may be bonded together to form long chains called polysaccharides.

Starch and glycogen- These are polysaccharides that function to store energy. They are

composed of glucose monomers bonded together producing long chains. Animals store extra

carbohydrates as glycogen in the liver and muscles. Between meals, the liver breaks down

glycogen to glucose in order to keep the concentration of glucoses in the blood. After meals, as

glucose levels in the blood rise, it is removed from the blood and stored as glycogen. Plants

produce starch to store carbohydrates. Amylopectin is a form of starch that is very similar to

glycogen It is branched but glycogen has more branches.

Structure of glycogen or

starch

Cellulose and chitin- Cellulose and chitin are polysaccharides that function to support and

protect the organism. The cell walls of the plant composed of cellulose. The cell walls of fungi

are composed of chitin. Cellulose is composed of beta-glucose monomers; starch and glycogen

are composed of alpha-glucose.

Structure of cellulose.

The glucose monomers of chitin (N-acetyl glucosamine) have a side chain containing

nitrogen.

Cotton and wood are composed mostly of cellulose. Humans and most animals do not have

necessary enzymes to break the linkages of cellulose or chitin.

Lipids-Lipids are compounds that are insoluble in water but soluble in nonpolar solvents. Some

lipids function in long term energy storage.Lipids is also an important component of cell

membranes.Lipids make 18-25% of body mass in lean adults. Like, carbohydrates lipid contain

carbon, hydrogen, and oxygen. They do not have 2:1 ratio of hydrogen to oxygen. The

proportion of electronegative oxygen atoms in lipids is usually smaller than in carbohydrates, so

there are fewer polar covalent bonds. As a result most lipids are insoluble in polar solvents such

as water: they are hydrophobic. Lipoproteins are soluble because the proteins are on the outside

and the lipids are on inside. The lipids includes triglycerides (fats and oils),phospholipids,

steroids, eicosanoids and a variety of other lipids including fat-soluble vitamins (A,D,E and K)

and lipoproteins.

Fats and oils (Triglycerides)- A triglyceride consists of two types of building blocks, a single

glycerol molecule and three fatty acid molecules. Three fatty acids are attached by dehydration

reaction. The hydrolysis, breaks down a single molecule of triglyceride into three fatty acids and

glycerol.

Fatty acids have a long hydrocarbon (carbon and hydrogen) chain with a carboxyl (acid) group.

The chains usually contain 16 to 18 carbons. Glycerol contains 3 carbons and 3 hydroxyl groups.

It reacts with 3 fatty acids to form a triglyceride or fat molecule.

Fats are nonpolar and therefore they do not dissolve in water.

Saturated fat- These are the triglycerides containing saturated fatty acids. The saturated fatty

acids have no double bonds between carbons. Examples- Palmitic acid (C15H31 COOH), stearic

acid (C15H35COOH). Examples of saturated fats- butter, cocoa butter, coconut oil, meats, etc.

Monounsaturated fats: contain fatty acids with one double bond between two fatty acid carbon

atoms. Examples- olive oil, peanut oil, etc. These fats are thought to decrease the risk of heart

disease.

Poly unsaturated fats: contain more than one covalent bond between fatty acid carbon atoms.

Examples- corn oil, sunflower oil, soybean oil and fatty fish. These fats are believed to decrease

the risk of heart disease. Unsaturated fatty acids have at least one double bond. Each double bond

produces a “bend” in the molecule.

Molecules with many of these bends cannot be packed as closely together as straight molecules,

so these fats are less dense. As a result, triglycerides composed of unsaturated fatty acids melt at

lower temperatures than those with saturated fatty acids. For example, butter contains more

saturated fat than corn oil, and it is a solid at room temperature while corn oil is a liquid.

Phospholipids- Phospholipids have a structure like triglycerides, but contain a phosphate group

in place of the third fatty acid. The phosphate group is polar and therefore capable of interacting

with water molecule. These are amphipathic in nature, as they have both polar and nonpolar

groups.

Phospholipids form a bilayer in a watery environment. They arrange themselves so that the polar

heads are oriented toward the water and the fatty acid tails are oriented toward the inside of the

bilayer.

Steroids- Steroids differs from triglycerides. They have four rings of carbon atoms. Body cells

synthesize other steroids from cholesterol. Cholesterol has a large nonpolar region consisting of

the four rings and a hydrocarbon tail. The steroids of the body are cholesterol, estrogens,

testosterone, cortisol, bile salts, and vitamin D. These are also known as sterols because they also

have at least one hydroxyl group (-OH). Polar hydroxyl groups make sterols weakly

amphipathic.

Important functions of the steroids- 1.Cholesterol is needed for cell membrane

structure.2.Estrogen and testosterone are required for regulating sexual functions.3.Cortisol is

needed for maintaining the normal blood sugar level. 4.Bile salts are needed for lipid digestion

and absorption. 5.Vitamin D is related to bone growth.

Cholesterol Estradiol

Testosterone

Other lipids: Eicosanoids are lipids derived from a 20-carbon fatty acid called arachidonic acid.

The two subclasses of eicosanoids are the prostaglandins and the leukotries.

Other lipids also include fatty acids- which under go either hydrolysis to provide ATP or

dehydration synthesis to build triglycerides and phospholipids.

Waxes- Waxes are composed of a long-chain bonded to a long-chain alcohol. They form

protective coverings for plants and animals (plant surface, animal ears).

Proteins- Proteins are large molecules that contain hydrogen, oxygen and nitrogen. Some

proteins also contain sulfur. Proteins have complex structure. The proteins make up 12-18% of

body mass in a lean adult. Proteins have many roles in the body and are largely responsible for

the structure of the body tissues. Enzymes are proteins that speed up most biochemical reactions.

Antibodies are proteins that defend against invading microbes. Hormones are also proteinsthat

regulate homeostasis. Some important functions of proteins are listed below.

Amino acids: These are building blocks of proteins. Each has a carboxylic group (COOH) and

an amino group (NH2).

A peptide bond is formed between two amino acids (NH2 and –COOH), a water molecule is

removed. This reaction is dehydration. The protein digestion is hydrolysis reaction. When two

amino acids combine, a dipeptide is formed. Adding another amino acid to dipeptide produces a

tripeptide. Further additions of amino acids result in the formation of a chain like peptide (4-9

aminoacids) or polypeptides (10-2000 or more amino acids). Small proteins consist of a single

polypeptide chain with about 50 amino acids. Larger proteins have hundreds or thousands of

amino acids and may consists of two or more polypeptide chains folded together.

Structure of proteins: The large number of charged atoms in a polypeptide chain facilitates

hydrogen bonding within the molecule, causing it to fold into a specific 3-dimensional shape.

The 3-dimensional shape is important activity of a protein.

Primary structure: The primary structure of a protein is the sequence of aminoacids that are

linked by covalent peptide bonds to form polypeptide chain. The two ends are N-terminal (NH2)

and C- terminal (COOH).

Secondary structure- The secondary structure is stabilized by hydrogen bonds. There are two

types secondary structure in proteins, the α-helix and β-pleated sheet. The α-helix is held

together by hydrogen bonds between the hydrogen and oxygen atom. Examples- keratin protein

present hairs, nails and skin. In β-pleated sheet, the hydrogen bond is formed between two chains

of the amino acid chains. Example- Fibroin- spider protein.

Tertiary structure- It refers to the overall- 3-dimesional shape of the polypeptide chain. Several

bonds are present in tertiary structure. The strongest S-S covalent bond between the sulfhydryl

groups of two monomers of the aminoacid cysteine. Many weak bonds –hydrogen bonds, ionic

bonds, and hydrophobic interactions also help for the folding pattern of the proteins. These bonds

are very important in maintaining the tertiary structure of some proteins.

Quarternary structure- Some proteins contain two or more polypeptide chains that associate

to form a single protein. These proteins have quaternary structure. For example, hemoglobin

contains four polypeptide chains.

Denaturation – This occurs when the normal bonding patterns are disturbed causing the shape

of the protein to change. This can be caused by changes in temperature, pH or salt concentration.

For example, acid causes milk to curdle and heat (cooking) causes egg white to coagulate

because the proteins within them denature.

Other kinds of proteins- Simple proteins contain only amino acids. Conjugated proteins

contain other kinds of molecules. For example- glycoproteins, nucleoproteins contain nucleic

acids, and lipoprotein contains lipids.

Nucleic acids- It is a macromolecule with acid property and it was isolated from the nucleus of

cells and hence it is named as nucleic acid. It is made up of C, H, O, N and P. Nucleic acids are

found in all organisms such as plants, animals, bacteria and viruses. They are found in the

nucleus as well as in the cytoplasma. It is a long chain polymer. It is composed of monomeric

units, called nucleotides. Each nucleotide consists of a nucleoside and a phosphate group. Each

nucleoside consists of a pentose sugar and a nitrogenous base. The sugar is ribose in case of

RNA and deoxyribose in case of DNA.

The nitrogenous bases are of two types, namely purine and pyrimidine. There are two main

purine bases, adenine and guanine. There are three pyrimidine bases. They are cytosine, thymine

and uracil. Cytosine and thymine are found in DNA. Cytosine and uracil are found in RNA.

Nucleosides: A base combined with a sugar molecule is called a nucleoside. In DNA four

nucleosides are present. They are adenosine, guanosine, cytidine and thymidine. In RNA

deoxyribose is replaced by ribose and the base thymine is replaced by uracil.

Nucleotides: A nucleotide is derived from a nucleoside by the addition of a molecule of

phosphoric acid. The DNA contains four different types of nucleotides. They are adenylic acid,

guanylic acid, cytidylic acid and thymidylic acid. The RNA contains uridylic acid instead of

thymidylic acid.

Polynucleotide: a number of nucleotide units linked with one another to form a polynucleotide

chain or nucleic acid.

Nucleic acids are classified into DNA and RNA. The RNA further classified into mRNA, tRNA

and rRNA.

Deoxyribonucleic acid: (DNA): It is the molecule of heredity. It functions as the genes.DNA is

present in all cells except plant virus. In eukaryotic cells, DNA is present in the chromosomes of

nucleus. In addition, the mitochondria and plastids contain DNA. In eukaryotic nucleus, the

DNA is in the form of a double helix. In bacteria, mitochondria and plastids the DNA molecules

are circular. In viruses and bacteriophages they are coiled. The number of DNA molecules in

eukaryotic cells corresponds to the number of chromosomes per cell. DNA is made up of 3

chemical components, namely 1.Sugar, 2.Phospharic acid and 3. Nitrogenous bases.

1.Sugar:

The sugar present in the DNA is called deoxyribose. It is a pentose sugar which contains five

carbon atoms (C5H10O4). It contains one O atom less than the ribose sugar. At carbon No.2 of

deoxyribose, is present a H-C-H group. But in ribose sugar the second carbon atom contains H-

C-OH group.

2.Phosphoric acid: (H3PO4)-

Deoxyribose sugar molecule linked with one phosphate group at 5th

position and another

phosphate group is linked with 3rd

position. This forms phosphate diester bond. This bond links

carbon 5’ in one nucleoside with carbon 3’ in the next nucleoside.

3.Nirogenous bases: These are N2 containing organic compounds. They are of two types,

namely purines and pyrimidines.

Purines: Purines are two – ringed N2 compounds. They are of two types, namely adenine and

guanine.

Pyrimidines: These are single ringed N2 compounds. They are two types, namely thymine and

cytosine. Thymine and cytosine are the pyrimidine bases of DNA.

Structure of DNA (Watson and Crick model):

In 1933 Watson and Crik designed the structure of DNA. It is called the Watson and Crick model

of DNA. They were awarded with Nobel Prize in 1962 for this work. According to them DNA is

in the form of double helix. DNA is the deoxy ribonucleic acid. It is a nucleic acid. It is made up

of two chains. Each chain is the polynucleotide chain. Each polynucleotide is made up of many

small units called nucleotides. Each nucleotide is made up of three chemical components,

namely a phosphoric acid, a deoxiribose sugar and a nitrogen base. The nitrogen bases are

adenine, guanine, thymine and cytosine.

The nucleotides of DNA are named according to the type of nitrogen bases present. As there are

four types of nitrogen bases, DNA contain four types of nucleotides, namely

1.AMP- Adenosine monophosphate (adenylic acid)

2.GMP- Guanosine monophosphate (guanylic acid )

3. TMP- Thymidine monophosphate (thymodylic acid)

4. CMP- Cytidine monophosphate (cytidylic acid)

In each nucleotide, the deoxyribose sugar is attached to a phosphoric acid at one side and a

nitrogen base at the other side. The phosphoric acid is linked to the sugar. The nitrogen base

molecule is joined to the sugar by a glycosidic bond. This bond is formed between sugar and

nitrogen base.

Many nucleotides are linked together to form a polynucleotide chain. Two nucleotides are joined

by a phosphodiesterase bond. It is formed between sugar of one nucleotide and phosphate

component of another nucleotide.

The linking between purines and pyrimidines is brought about by hydrogen bonds. There are two

hydrogen bonds between A and T (A=T), and 3 hydrogen bonds between G and C (G= C). The

amount of adenine is equivalent to the amount of thymine and the amount of guanine is

equivalent to the amount of cytosine. The two chains of a DNA are complimentary to each other.

At one end of the polynucleotide chain, the 3rd

carbon atom of the sugar is free and it is not

linked to any nucleotide. This end is called 3 prime (3’) end. At the other end of the 5th

carbon of

the sugar is free and this end is called 5prime (5’) end.

DNA strand is antiparallel as they run in opposite direction. The DNA molecule is in the form of

a double helix. The two polynucleotide chains are coiled around each other to form a double

helix. The width (diameter) of DNA is 20A0. The DNA has two external grooves, namely major

groove and minor groove. The major groove is wider and deep. The minor groove is narrow. The

distance between two nucleotides is 3.4 A0.

Properties of DNA;

1. Size of the DNA molecule-The size of the DNA molecule varies from organism to organism.

It depends upon the size of the chromosome and the number of chromosomes found in each

living cell. The size basically depends upon the number of nucleotides present in each DNA

molecule. The size of DNA molecule ranges from 0.7 µm to 40,000mm (4cms).

2. Fragility of DNA molecule: The DNA molecule is highly fragile. Smaller DNA can be

isolated without any damage, but large sized DNA (above 2X 108 deltons) undergoes breakage

during their extraction.

3. Denaturation: Denaturation refers the separation of the two strands of a DNA. Denaturation

is brought about by high temperature, acid, or alkali. During denaturation there is breakdown of

hydrogen bonds between base pairs. Since G-C base pairs have 3 hydrogen bonds and A-T pairs

have 2 hydrogen bonds, G-C base pairs are more stable and it needs more temperature for

denaturation.

4.Renaturation: The denatured single stranded DNA can be made into double stranded DNA by

cooling or by neutralizing the medium. This process is called denaturation.

5.Effect of pH on DNA: The DNA is stable around the neutral pH in the solution. Further

increase in pH (alkali treatment) causes stand separation and finally denaturation occurs.

6.Stability : The DNA is a highly stable molecule. The stability is due to two forces.

a.( Hydrogen bonding between the bases. b.) Hydrophobic interactions between the bases.

Hydrogen bonds

7.Hyper chromic effect: DNA molecule absorbs light energy. This is a property of individual

bases. The intact DNA absorbs less light energy as its bases are packed into a double helix. A

denatured DNA molecule absorbs more light as its bases in single strands are exposed.

Functions of DNA: It plays an important role in all biosynthetic and heriditory functions of all

living organisms.

1.It acts as the carrier of genetic information from generation to generation.

2. DNA is a very stable macromolecule in almost all living organisms.

3.It controls all developmental processes of an organisms and all life activities.

4.DNA synthesizes RNAs.

5. DNA is the genetic code which is responsible for protein synthesis.

Nucleotides: Nucleotides are defined as phosphoric acid esters of nucleosides. A nucleotide is

made up of 3 components, namely a nitrogen base, a pentose sugar and a phosphoric acid.

The nucleotides are named according to purines and pyrimidines. AMP, GMP, TMP,CMP and

UMP. In addition many nucleotides occur freely in the tissues. They are ADP and ATP. On

hydrolysis nucleotide splits into phosphoric acid and a nucleoside. The nucleoside is made up of

a base and a pentose sugar. The hydrolysis of ATP yields ADP and energy.

Biological significance of nucleotides:

1. Nucleotides form the main components of nucleic acids.

2. Genetic material: Deoxyribonucleotides of DNA transmit hereditary characters from parents

to offspring.

3. Nucleotides functions as the source of high energy.Eg ATP, UTP, CTP, etc.

4. ATP is involved in oxidative phophorylation.

5. Certain nucleotides function as coenzymes. Eg UDPG, CoA, FMN, FAD.

6. Certain nucleotides function as vitamin B. Eg. FMN, FAD, NAD. Etc.

Nucleosides: Compounds that contain nitrogen bases linked to pentose sugars are called

nucleosides. There are two main types: ribonucleoside and deoxyribonucleoside. There are 5

types of nucleosides- adenosine, guanosine, thymidine, cytidine, uridine.

Types of DNA: DNA is classified into various types:

1.Double stranded DNA: It is also called as double helical DNA. In most of the organisms

except a few viruses, the DNA has a double stranded structure.

2.Single stranded DNA: Eg. Some viruses, E.coli, extra chromosomal satellite.

3. A-DNA: It is a double helical DNA having 11 residues per turn. It has a right handed helix. It

is formed by the dehydration of B.DNA.

A, B and Z DNA

4.B-DNA: This is the Watson and Crick double helix having 10 residues per turn. It is also right

handed. 5.Z- DNA: It is the left handed double helix having 12 residues per turn.

6. Circular DNA: It is circular in shape. It is found in bacteria, virus, mitochondria and

chloroplast. The circular DNA may be single stranded or double stranded. Single stranded

circular DNA is found in some viruses. Double stranded circular DNA is found in bacteria,

viruses, mitochondria, chloroplast, etc.

7. Relaxed DNA: Circular DNA without any helical coiling is called relaxed DNA.

8. Supercoiled DNA: It is supercoiled DNA. It can produce negative super coiling and positive

supercoiling. The degree of supercoiling is controlled by topoisomerases and gyrases.

9.Palindromic DNA: A double helix is formed by two paired strands of nucleotides that run in

opposite directions in the 5I- to 3I sense and the nucleotides always pair in the same way A-T for

DNA , with Uracil (U) for RNA, Cytosine ( C ), a nucleotide said to be palindrome if it is equal

to its reverse complement. For example, the DNA sequence ACCTAGGT is palindromic

sequence because its compliment is TGGAGGT. The sequence of nucleotide goes in one

direction and in another direction in the second strand.

10. Repetitive DNA or satellite DNA: When very short sequences of base pairs are repeated

many times in DNA, the DNA is called repetitive DNA or satellite DNA. All eukaryotes, except

yeast, contain repetitive DNA. Repetitive DNA is absent in prokaryotes. The repetitive DNA can

replicate but cannot transcribe mRNA for protein synthesis. Repetitive DNA is therefore inert.

Ribonucleic acid (RNA): It is a nucleic acid containing ribose sugar. It is found in large amount

in the cytoplasm and at a lesser amount in the nucleus. In the cytoplasm it is found mainly in the

ribosomes and in the nucleus it is mainly found in the nucleolus. RNA is formed of a single

strand. It consists of several units called ribo-nucleotides. Hence each RNA molecule is formed

of several nucleotides. Each nucleotide is formed of different molecules, namely phosphate,

ribose sugar and nitrogen base. The nitrogen bases are purines and pyrimidines. The purine bases

present in the RNA are adenine and guanine. The pyrimidines present in the RNA are cytosine

and uracil. The RNA molecule is normally single stranded, sometimes the stand may be folded

back upon itself and this double strand may be coiled to form a helical structure like that of

DNA. In RNA purines and pyrimidines are not present in equal amount.

RNA structure

There are three types of RNA. They are mRNA, tRNA and rRNA

mRNA: This type of RNA carries genetic information for protein synthesis from the DNA to the

cytoplasm. The mRNA forms about 3 to 5% of the total cellular RNA. The mRNA is synthesized

as a complimentary strand upon the chromosomal DNA. The genetic message from DNA is

transcribed in this hybrid mRNA. The mRNA carries the message in the form of triplet codes.

The hybrid mRNA inside the nucleus is called heterogenous nuclear RNA (hnRNA). It is

processed in the nucleus and enters the cytoplasm through nuclear membrane. In the cytoplasm

mRNA are deposited on some ribosomes. In the ribosomes mRNA acts as a template for protein

synthesis.

The life span of mRNA in bacteria is about 2 min. In eukaryotes it lives for few hours to a few

days.In the animal eggs and a plant seed, the mRNA is stabilized for months or years. Protein

synthesis must be carried within this life span.

Types of mRNA: There are two types of mRNA

a)Monocistronic mRNA: It is formed from a single cistron (functional gene). It contains the

genetic information to translate only a single protein chain (polypeptide). The eukaryotic mRNAs

are monocistronic.

b) Polycistronic mRNA: It is formed from several cistrons. The polycistronic mRNA carries

several open reading frames (ORFs), each of which is translated into a polypeptide. Hence

polycistronic mRNA translate several polypeptide chains. The prokaryotic mRNA are

polycistronic.

Structure of mRNA: It is a single stranded polynucleotide chain. Each nucleotide is made up of

many nucleotides. Each nucleotide contains a phosphoric acid, a ribose sugar and a nitrogenous

base. The nitrogenous base may be adenine or guanine or cytosine or uracil.Among RNAs,

mRNA is the longest one. Most of the mRNAs contain 900 to 15000 nucleotides. One end of the

mRNA is called 5’ end and other end is called 3’ end. At the 5’ end a cap is found in most

eukaryotes and animal viruses. The cap is formed by the condensation of the guanylate residue.

The cap helps the mRNA to bind with ribosomes.

mRNA

The cap is followed by a non-coding region1 (NC1). It does not contain code (message) for

protein and hence it cannot translate protein. It is formed by 10 to 100 nucleotides and is rich in

A and U residues. The non-coding region is followed by the initiation codon. It is made up of

AUG. The initiation codon is followed by the codon region which contains code for protein. It

has an average 1,500 nucleotides. The codon region is followed by a termination codon. It

completes the translation. It is made up of UAA or UAG or UGA in eukaryotes. The termination

codon is followed by non-coding region 2(NC2). It has a nucleotide sequence of AAUAAA. At

the 3’ end of mRNA, there is a polyadenylate sequence (poly A). It consists of 200 to 250

adenylate nucleotides (AAAAA….). But as the age increases, the poly A shortens.

The mRNA is synthesized from a DNA strand through the action of an enzyme called RNA

polymerase. The synthesis of mRNA is called transcription. mRNA carries the genetic

information from the DNA. The code decides the type of protein to be synthesized.

The mRNA carries genetic information from DNA. The genetic information carried by the

mRNA is called genetic code. The genetic code is the sequence of nitrogen bases in mRNA. The

genetic code is formed of several codons. Each codon is a sequence of three nitrogen bases

which codes for one aminoacid. As each codon is formed of threenucleotides, it is called a triplet

code. Each mRNA contains the codons for one polypeptide chain. It the mRNA contains 900

nucleotides the polypeptide chain synthesized by this mRNA will contain 300 aminoacids.

Transfer RNA (tRNA): It is a ribonucleic acid which transfers the activated aminoacids to the

to the ribosomes to synthesize proteins. It is so small that it remains in the supernatant during

centrifugation. Hence it is also called soluble RNA or supernatant RNA. It serves as an adaptor

molecule to attach amino acids. Hence tRNA is also called adaptor RNA. It constitutes 10 to

15% of the total weight of RNA of the cell. It has a molecular weight of 25000 to 30000 and a

sedimentation co-efficient of 3.8S.

Structure of tRNA: The tRNA is made up of 73 to 95 nucleotide units called ribonucleotides.

Each nucleotides unit is made up of three components, namely a phosphate, a ribose sugar and a

nitrogenous base such as adenine, guanine, cytosine or uracil. The tRNA is in the form of single

polynucleotide chain having 3’ and 5’ ends. The polynucleotide chain of tRNA is folded on itself

and attains the shape of clover leaf. The 3’ and 5’ ends of tRNA lie side by side as a result of

folding. The 3’ end always ends in CCA base sequence. This is the site for the attachment of

activated amino acid. The 5’ end terminates in G or C. The t RNA has 5 arms.

a.Amino acid acceptor arm, b) D-arm, c) Anticodon arm, d)Variable arm, e) T ΨC arm. Each

arm is made up of a stem and a loop. But the acceptor arm has no loop; the variable arm has no

stem. In the stem, the bases pair with each other (A-U and G-C). There is no base pairing in the

loops.

a) Amino acid acceptor arm: In the amino acid acceptor arm, the stem does not end with loop.

The acceptor has 3’ end of the nucleotide chain. The terminus of the acceptor site has a constant

CCA base sequence. To this base amino acids are attached to form aminoacyl tRNA. The 5’ end

of the arm comes near the 3’ end due to folding. Its terminus is either G or C.

b) D.arm: The D arm has 3 to 4 bases in the stem and 7 to 11 unpaired bases in the loop. The

loop is called dihydrouridine loop (DHU) or D-loop.

c)Anticodon arm: In the anticodon arm, the stem has 5 paired bases and the loop has 7 unpaired

bases. The loop is called anticodon loop. Three of the 7 unpaired bases in the loop (anticodon)

determine the pairing of tRNA with the specific codon of mRNA.

d) Variable arm: In variable arm the stem may or may not be formed. The variable arm or mini

arm has a loop with 4-5 bases.

e) T ΨC arm: The T ΨC arm contains a constant T ΨC sequence. Its loop has ribosome

recognition site.

The tRNA molecules are named according to the amino acid to which it gets attached. For

example, tRNA carrying alanine can be called tRNAal. The tRNA molecules are synthesized, at

particular regions of DNA by a process called transcription. About 40 to 80 genes or cistrons are

involved in tRNA transcription.. The hybrid tRNA has base sequences complimentary to the

mother DNA in the beginning. But, after the completion of transcription, the nitrogenous bases

are altered at certain points in the nucleotide chain.

Functions of tRNA: tRNA picks up a specific activated amino acid from the amino acid pool in

the cytoplasm (aminoacyltRNA). The amino acid is then transferred to the ribosome in the

cytoplasm where the proteins are synthesized. The attachment with ribosome depends upon the

codes in the mRNA and anticodons in the tRNA. Finally it transmits its amino acid to the new

polypeptide chain.

Ribosomal RNA (rRNA): It is a ribonucleic acid present in the ribosomes and hence it is called

ribosomal RNA. It is also called insoluble RNA. It constitutes about 80% of the cellular RNA. It

is formed by a single strand. It is a polynucleotide chain. Each strand is formed of many

nucleotide units. Each nucleotide is formed of three components- a phosphare, a ribose sugar and

a nitrogen base. The purine bases present in rRNA are adenine and guanine and the pyrimidine

bases are cytosine and uracil. Each strand has a 5’ end and a 3’ end. In some regions, the single

strand is twisted upon itself to form a double helix. In the helical regions most of the base pairs

are complimentary. They are joined by hydrogen bonds. In the unfolded single stranded regions,

the base pairs are not complimentary. In prokaryotes, the rRNA has 3 types: 23S, 5S, and 16S. In

the mammals, 4 types of rRNA have been found: 28S, 5.8 S, 5S and 18S. The unit S strand for

Svedberg, which is a measure of the sedimentation rate. After rRNA molecules are produced in

the nucleus, they are transported to the cytoplasma, where they combine with specific proteins to

form a ribosome. In prokaryotes, the size of a ribosome is 70S, consisting of two subunits: 50S

and 30S. The size of a mammalian ribosome is 80S, comprising a 60S and a 40S subunit.

Proteins in the larger subunit are designated as L1, L2, L3, etc.(L=large). In the smaller subunit,

proteins are denoted by S1, S2, S3, etc.

Composition of rRNA

During protein synthesis, the ribosome binds to mRNA and tRNA as above. Only the tRNA

containing the anticodon which matches mRNA codon may join the complex.

Functions of rRNA; It forms the main bulk of the cytoplasmic RNA. Its function is not clearly

known. However it is believed that rRNA plays the major role in protein synthesis.

c) DNA replication: General, bacterial and eukaryotic DNA replication. Replication is the duplication of DNA. By replication DNA produces exact copies of its own

structure. Replication occurs inside the chromosomes. It occurs during interphase. The parent

DNA strands function as templates for the synthesis of new DNA strands. New DNA is produced

by semi conservative process. Of the two strands produced, one strand is the parental strand and

the second strand is newly synthesized.

Replication starts at a specific point called origins. At this origin, the two strands are separated.

This separation is brought about by the enzyme helicase. At the point where the two strands are

separated, a replication fork is formed. The fork appears in the form of Y. The duplication of

DNA is brought about by the movement of the replication fork. Single stranded DNA binding

proteins or SSB, binds to single-stranded regions of DNA to prevent premature annealing, to

prevent the single-stranded DNA from being digested by nucleases and allow other enzymes to

function effectively upon it.

Leading strand- In one DNA strand, the daughter strand is synthesized as a continuous strand.

This strand is called leading strand because it is synthesized first. The leading strand is

synthesized continuously in the 5’ 3’ direction by DNA polymerase.

DNA replication Bidirectional replication

On the leading strand, a DNA polymerase “reads” the DNA and adds nucleotides to it

continuously. This polymerase is DNA polymerase III in prokaryotes. In human cells the leading

and lagging strands are synthesized by Pol α and Pol δ within the nucleus and Pol γ in the

mitochondria.

RNA primer- A primer is a strand of nucleic acid (10-12 nucleotides) that serves as a starting

point for DNA synthesis. They are required for DNA replication because the enzymes that

catalyze this process, DNA polymerases can only add new nucleotides to an existing strand.

The polymerase starts replication at the 3’ end of the primer and copies the opposite end. In most

cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand

of RNA. The RNA primer is synthesized by DNA dependent RNA polymerase (primase). The

RNA primer is degraded at the end of DNA replication.

Lagging strand- In the second DNA strand, the daughter strand begins slightly later. Hence this

daughter strand is called lagging strand. The lagging strand is synthesized in short fragments

called Okazaki fragments. The replication is semi conservative discontinuous because the DNA

is synthesized in short segments. The enzyme DNA ligase joins the Okazaki fragments into a

long polynucleotide chain. DNA ligases catalyze formation of a phosphodiester bond between

the 5’ phosphate of one strand of DNA and the 3’ hydroxyl of another.

Replication may occur in one direction from the point of origin or in both directions. When

replication occurs in only one direction, it is called unidirectional replication. When replication

occurs in both directions, it is called bidirectional replication.

Bacterial (prokaryotic) DNA replication- DNA replication in E.coli begins at a single, defined

DNA sequence of 245 base pairs called oriC . A protein called DnaA gets concentrated near oriC

and get complexes with ATP. This complex binds with specific 9-bp repeats at oriC. These

distors the DNA, leading to the opening of adjacent 13- bp repeats in the DNA. The opened

DNA strand allows protein complex to enter the DNA bubble. The protein complex consists of

DNA helicase (Dna-B) and a DNA helicase loader (Dna-C). DNA helicase loader open the

DNA helicase protein rings and place the rings around the single stranded DNA. The loaders are

then released. The helicases use energy from ATP hydrolysis to unwind the DNA helix at each

of the two replication forks. Each DNA helicase recruits an enzyme called DNA primase,(DNA

dependent RNA polymerase) which synthesizes RNA primer on the DNA template. The RNA-

primer contains 10-12 nucleotides.

The main replication enzyme in E.coli is called DNA polymerase III. This enzyme synthesizes

the new DNA 5’ to 3’ direction. This is leading strand. In contrast, the other new strand called,

called the lagging strand is built in fragments called, Okazaki fragments. The template strands

are antiparallel, with their 3’ and 5’ ends oriented in opposite directions. Many proteins

participate in DNA replication. Single- strand DNA binding proteins quickly coat the exposed

single stranded DNA. These proteins protect the DNA strand against nucleases. DNA replication

continues as the DNA polymerase on the lagging strand meets the 5’ end of the next primer.

After the DNA helicase has moved approximately 1000 bases, a second RNA primer is

synthesized at the fork. The cycle continues for the length of the template strands. The lagging

strand consists of Okazaki fragments with a segment of RNA at one end. The RNA is cleaved by

an enzyme called RNase H. Another enzyme DNA polymerase I fills the large gap between

Okazaki fragments. Finally DNA ligases catalyze formation of a phosphodiester bond between

the 5’ phosphate of one strand of DNA and the 3’ hydroxyl of another. During replication, DNA

can become super coiled. DNA gyrase (topoisomerase II) prevents this super coiling.

Topoisomerase IV separates the newly formed daughter circular DNA strand from the parent.

Eukaryotic DNA replication: DNA replication in eukaryotes is much complicated in

prokaryotes, although there are many similar aspects. Eukaryotic cells only initiate DNA

replication at a specific point in the cell cycle- the beginning of the S phase. However, pre-

initiation occurs in the G1 phase.

d) The cell cycle: Restriction point, cell cycle regulators and modifiers.

Cell cycle: (cell-division cycle) It is the series of events that takes place in a cell leading to its

division and duplication (replication). In prokaryotic cells, the cell cycle occurs via a process

termed binary fission. In eukaryotic cells, the cell cycle can be divided in two periods- a)

interphase, b) mitosis

a) Interphase- During this phase the cell grows, accumulating nutrients needed for mitosis and

duplicating its DNA.

b) Mitosis (M)-phase- During which the cell splits itself into two distinct cells

The duration of the cell cycle varies from hours to years. For example, the cell cycle of

Paramecium aurelia has duration of 6h. A typical human cell has duration of 90h.

a)Interphase: It is the longest phase. In a typical human cell, out of the 90h, interphase lasts for

89h.

Resting (Go phase): In interphase the cell prepares itself to cycle. The term post-mitotic is

sometimes used to refer both quiescent and senescent cells. Non-proliferative cells in eukaryotes

generally enter the quiescent Go state from G1 and may remain quiescent for longer period of

time or indefinitely (e.g.cardiac cells and neurons). In multicellular eukaryotes, cells enter the Go

phase from the G1 phase and stop dividing. Some cells enter the Go phase semi-permanently, e.g.

some liver and kidney cells.

Characters of interphase: It is the resting phase of the cell. Resting refers to the rest from

division. But, the cells in the interphase are metabolically active. The metabolic activities are

high in this phase. The cell grows during phase. During this phase mRNA and rRNA are

synthesized. Throughout the interphase the chromosomes are extended and are not visible in the

light microscope. The chromosomes duplicates into two chromatids. The centrioles duplicates

into two. Thus two centrioles are formed. The centrospheres of centrioles, microtubules arise.

These microtubules form asters.

Stages of interphase: Interphase consists of 3 sub-stages. They are G1 phase, S phase and G2

phase.

G1 Phase: G stands for gap. It is the first phase within the interphase, from the end of the

previous M phase. It is also called the growth phase. This phase is the gap period between a

mitotic phase and the S phase of the cycle. This period starts immediately after division. The

daughter cells grow and increase in size during this phase. It is a longer phase. It lasts for even

years. The nerve cells remain permanently in G1 phase. Generally, this stage lasts for 25 to 50%

of the total interphase. During this phase 20amino acids are formed, from which millions of

proteins and enzymes are formed, which are required in S phase. During this phase mRNA,

rRNA and tRNAs are formed. During this phase new cell organelles are formed.

G1 phase consists of four sub-phases: Competence (g1a), entry (g1b), progression (g1c) and

assembly (g1d). These sub-phases may be affected by limiting growth factors, nutrient supply,

temperature, and additional inhibiting factors. A rapidly dividing human cell which divides every

24h spends 9h in G1 phase. The DNA in a G1 diploid eukaryotic cell is 2n, meaning there are two

sets of chromosomes present in the cell. The genetic material exists as chromatin.

There is a restriction point present at the end of G1 phase. Signals from extracellular growth

factors are transducer in a typical manner. Growth factors bind to receptors on the surface.

Accumulation of cyclin D’s is essential. Cyclins are a family of proteins that control the

progression of cells through the cell cycle by activating cyclin-dependent kinases (Cdk)

enzymes. Cyclin D acts as a mitogenic signal sensor.

S-phase: S stands for synthesis: During this phase DNA synthesis occurs. The DNA molecule

duplicates. All the chromosomes have been replicated. This period lasts for 35 to 40% of

interphase. During this phase, synthesis is completed as quickly as possible due to the exposed

base pairs may be destroyed by the external proteins (drugs) or any mutagens (such as nicotine).

Cyclins, when bound with the dependent kinases such as Cdk1 proteins form the maturation-

promoting factor (MPF). MPFs activate other proteins through phosphorylation. These

phosphorylated proteins, in turn, are responsible for specific events during cell division such as

microtubule formation and chromatin remodeling.

DNA damage checkpoints: These sense DNA damage both before the cell enters S phase (a G1

checkpoint) as well as after S phase (a G2 checkpoint). Damage to DNA before the cell enters S

phase inhibits the action of Cdk2 thus stopping the progression of the cell cycle until the damage

can be repaired. If the damage is so severe, that it cannot be repaired, then the cell destructs by

apoptosis. Damage (UV radiation, oxidative stress, etc) to DNA after S phase (the G2

checkpoint), inhibits the action of Cdk1 thus preventing the cell from proceeding from G2 to

mitosis. In the S phase if DNA replication stops at any point on the DNA, the progress through

the cell cycle is halted until the problem is solved.

G2 phase (pre-mitotic phase) The G2 phase is the gap period between S-phase and mitotic (M)

phase of a cell cycle. It is the second growth phase. It is a period of rapid cell growth and protein

synthesis which the cell readies itself for mitosis. The nucleus increases in volume. Metabolic

activities essential for cell division, occur during this phase. mRNA, tRNA and rRNA synthesis

also occur. It is not a necessary part of the cell cycle. The G2 phase is followed by mitotic phase.

b)M-phase (Mitotic phase): This is the division phase. During this phase the cell divides. This

phase has a short duration. A typical human cell cycle has duration of 90h. Of these the M phase

has duration of 45 to 60min. This phase has two sub-phases called karyokinesis and cytokinesis.

Karyokinesis refers to the cell division of nucleus into two daughter nuclei. It has 4 sub-stages,

namely prophase, metaphase, anaphase and telophase, Cytokinesis refers to the cell division of

the cytoplasm resulting in two daughter cells.

Cell cycle checkpoints (Restriction points): These are the cell cycle control mechanisms in

eukaryotic cells. These checkpoints verify whether the processes at each phase of cell cycle have

been accurately completed before progression into the next phase. There are three main

checkpoints that control the cell cycle in eukaryotic cells. They are

1.G1 checkpoint (G1restriction point)

2.G2 checkpoint

3.Metaphase checkpoint.

1.G1 checkpoint (G1restriction point): This checkpoint is present at the end of the G1 phase

and just before of the S phase of the cell cycle. This checkpoint helps in taking the decision of

whether the cell should divide, delay division, or enter a resting stage (Go phase). If there are

unfavorable conditions for the cell division, then this restriction point restrict the progression to

the next phase by passing the cell to Go phase for an extended period of time.

This restriction point is controlled mainly controlled by the action of the CKI-p16 (CDK

inhibitor p16). The inhibited CDK not bind with cyclin D1, hence there is no cell progression.

The E2F group of genes (transcription factors) that are present in eukaryotic cells, they control

the progression of cell cycle. Transcription activators such as E2F1, E2F2, and E2F3a promote

the cell cycle, while repressors like E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8 inhibit the cell

cycle. This restriction point is overcome by the increased expression of cyclin D, which then

interact with CDK4/6 phosphorylate the tumour suppressor retinoblastoma (Rb), which relieves

the inhibition of the transcription factor E2F. E2F is then able to cause expression of cyclin E,

which then interacts with CDK2 to allow for G1-S phase transition. This brings the cell to the end

of the first checkpoint.(unphosphorylated Rb inhibits the E2F).

2.G2 checkpoint: This restriction point is located at the end of the G2 phase. This checks the

number of factors which are essential for the cell division. Maturation-promoting factor or

mitosis promoting factor or M-phase promoting factor- (MPF) is a protein composed of cyclin-B

and CDK-1. This protein promotes the G2 phase into the entrance of M-phase. MPF is activated

at the end of G2 by a phosphatase (Chk) which removes an inhibitory phosphate group added

earlier.

The main functions of MPF in this restriction point are

a.Triggers the formation of mitotic spindle.

b.Promotes chromosome condensation.

c.Causes nuclear envelop breakdown.

If there are any damages are noticed in this restriction point, then phasphatase not activate the

MPF, resulting in the arrest of cell cycle in G2 phase till the repair of the damaged DNA. This

prevents the transfer of defected DNA into the daughter cells.

3.Metaphase checkpoint: This occurs at metaphase. Anaphase-promoting complex (APC)

regulates this checkpoint. This is also called spindle checkpoint. This checks whether all

chromosomes are properly attached to the spindle or not. This also governs the alignment of the

chromosomes and integrity of the spindles. If there are mistakes then it delays the cell in entering

into anaphase from metaphase.

Cell cycle regulators: The cell cycle is regulated by cycles, cyclin-dependent kinsases (CDKs),

and cyclin-dependent kinase inhibitors (CDKIs).

1.Cyclins: Their concentration varies during the cell cycle. Cyclins are the family of proteins

which regulates the cell cycle. There are several types of cyclins that are active in different parts

of the cell cycle and causes phosphorylization of CDK. There are also several”orphan” cyclins

for which no CDK partner has been identified. For example, cyclin F is an orphan cyclin that is

essential for G2/M transition. There are two main groups of cyclins:

a.G1/S cyclins: Examples- Cyclin A,D and E. These cyclins are essential for the control of the

cell cycle at the G1/S transition. CyclinA/CDK 2- active in S phase. Cyclin A binds to S phase

CDK 2 and is required to progress through the S Phase. Cyclin A/CDK 2 is inhibited by the

complex p21CIP.

The un-phophorylated form of Rb binds with E2F family of transcription factors which controls

expression of several genes involved in cell cycle progression (example-cyclin-E). Rb acts as a

repression, so in complex with E2F it prevents the expression of E2F genes, and this inhibits the

cell progression from G1 to S phase. The binding of cyclin D/CDK4 and cyclin D/CDK 6 lead to

partial phosphorylation of Rb, by reducing its binding to E2F. The E2F gene activates the

expression of cyclin E bind with CDK 2 and causes complete phosphorylation of Rb. This

progresses the cell cycle from G1 to S phase.

a.G2/M cyclins: Example; cyclin B/ CDK1-. These are essential for the control of the cell cycle

at the G2/M transition. G2/M cyclins accumulate steadily during G2 and are abruptly destroyed as

cells exit from mitosis (at the end of the M-Phase). Cyclin B/ CDK1- regulates progression from

G2 to M Phase. Cyclin B is a mitotic cycin. The amount of cyclin B (which binds to CDK1) and

the activity of cyclin B-CDK complex rise through the cell cycle until mitosis. The cyclinB-

CDK1 complex is called maturation promoting factor (MPF). There are two types of cyclin B.

Cyclin B1/CDK1- This complex is involved in the early events of mitosis such as chromosome

condensation, nuclear envelop breakdown. This complex is localized in the microtubules.

Cyclin B2/CDK1- This complex is localized in the Golgi apparatus. Cyclin B2 is also binds to a

gene -transforming growth factor (TGF beta receptor 2). This gene complex transcribes several

genes required for the cell proliferation.

2.Cyclin-dependent kinsases (CDKs)- These are a family of protein kinases that regulates the

cell cycle. They are present in all known eukaryotic cells.

Phase Cyclin CDK

Go C CDK3

G1 D,E CDK4, CDK2, CDK6.

S A, E CDK2

G2 A CDK2, CDK1

M B CDK1

CDK levels remains relatively constant throughout the cell cycle. The four major mechanisms of

CDK regulation are cyclin binding, CAK phosphorylation, regulatory inhibitory phosphorylation

and binding of CDK inhibitory subunits.

c. Cyclin-dependent kinase inhibitors (CDKIs) – CDKI is a protein which inhibits cyclin-

dependent kinase (CDK). Cell cycle progression is negatively controlled by cyclin-dependent

kinases inhibitors (called CDIs, CKIs or CDKIs). These are involved in cell cycle arrest at the G1

phase.

Examples:

CDKI Interacting CDK CDKI Interacting CDK

p16 CDK4, CDK 6 p21 Cyclin E1 and CDK2

p15 CDK4 p27 CDK3, CDK 4, CDK 2, Cyclin E1

p18 CDK4, CDK 6 p57 Cyclin E1, CDK2

p19 CDK4, CDK 6

Cell cycle modulators: a.Flavopiridol- It is a flavonoid derived from an indigenous plant from

India a flavonoid derived from an indigenous plant from India. It is being developed by Aventis

oncology in collaboration with the national cancer institute (NCI). Its use is presently under

investigation for a variety of solid tumors as well as hematological cancers. It is a cyclin

dependent kinase (CDK) inhibitor of CDK1, CDK2, CDK 4 and CDK 6 at nano molar

concentrations, resulting in cell cycle arrest at both the G1/S and G2/M transitions.

b.Bryostatin- I. It transiently induces p21 (inhibitor protein) and causes subsequent

dephophorylation and inactivation of CDK2. Bryostatin also decreases expression of cyclin B,

preventing CDK 2 activation. The net effect is G2 phase arrest of the cell cycle.

c.UCN-01 is a protein kinase inhibitor under development as a novel anticancer drug. It has cell

cycle effects at both the g1/S and G2/M transitions. At G1/S, UCN-01` dephophrylates Rb and

causes the destruction of free E2F proteosome (it is a protein that digest endogenous proteins)

degradation and ubiquitination (protein is inactivated by attaching abiquitin- a small protein) .

The net effect is arrest in G1 and significant reduction in early S-phase proteins. At the G2/M

transition, through inhibition of Chk1 (a phosphatase which activates MPF), UCN-01 increases

the activity of cyclin B/CDK2, resulting in G2 checkpoint abrogation (abolition) and premature

entry into M phase.

b) Chromosome structure: Pro and eukaryotic chromosome structures, chromatin

structure, genome complexity, the flow of genetic information

Chromosomes: Chromosomes are self – producing thread like structures located inside the

nucleus. They are called (Chroma = color, soma = body) because they are easily stained with

dyes. They are the vehicles of the heredity. Then number of chromosomes varies from species to

species. But the number remains constant among the members of the same species. The lowest

number of chromosomes is 2 and it occurs in Ascaris megalocephala. The maximum number of

chromosomes is 1700 and it occurs in radiolarian (protozoa). The number of chromosomes in

some animals and plants given as follows.

Animals- 1).Ascaris megalocephala = 2, 2)Drosophilia= 8, 3)Toad= 22, 4.)frog= 26, 5) Rat=42,

6)Rabbit= 44, 7) Man= 46, 8)Gorilla= 48, 9)Cow=60. 10) Pigeon= 80, 12) Radiolarian= 1700.

Plants- 1) Pea= 14, 2) Onion=16, 3) Cabbage= 18,4) Tomato=24, 5) Potato =48, 6) Sugar cane=

80.

In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called

chromosomes. Each chromosome is made up of DNA tightly coiled many times around the

beads. Each bead is a nucleosome and consists of double stranded DNA wrapped twice around a

core of 8 protein called histones, which helps stabilizing the coil and folded structure of

chromosome. Chromosomes are not visible in the cell’s nucleus-not even under a microscope-

when the cell is not dividing. But during cell division the tightly packed chromosomes are visible

under a microscope. Each chromosome has a central constricted point called centromere, which

divides the chromosome into two sections, or arms. The short arm is “p arm” and the long arm is

“q arm”. Generally the chromosomes are arranged in pairs. A pair of similar chromosome is

called homologous chromosomes. The somatic cells contain two sets of chromosomes. This

number is called diploid number (2n). The gametes contain only one set of chromosomes. This

number is haploid number and it is represented as n. Sometimes a cell may contain more than

two sets of chromosomes. This number is celled polyploid (3n,4n,5n,etc).

Shape and size of the chromosomes: The shape of a chromosome is largely

determined by the position of its centromere. On this basis, chromosomes are classified into four

types.

1.Telocentric, 2.Acrocentric, 3.Sub-metacentric, 4.Metacentric.

1.Telocentric: The centromere is located at the end of the chromosome. Such chromosomes are

rare. It exists normally in certain species of protozoa. Human do not posses telocentric

chromosomes.

2.Acrocentric: These are rod like chromosomes having very small arm and a very long arm.

This is characteristic of locusts.

3.Sub-metacentric: These chromosomes are L shaped having unequal arms.

4.Metacentric: These chromosomes are V-shaped . They have arms of equal length. They are

characteristics of amphibian.

The size of the chromosomes ranges from 0.1 micron to 30 microns. The diameter varies from

0.2 micron to 2 microns. In general plants have larger chromosomes than animals. The plant

Trillium has chromosomes with length of 32 microns. The length of human chromosomes varies

from 4 to 6 microns. A typical somatic chromosome has an elongated cylindrical body with two

arms. It consists of a pellicle, matrix, chromonema, chromomeres, centromere or primary

constriction, secondary constriction, satellite bodies and telomeres.

Structure of chromosomes: A chromosome is usually composed of the following parts.

1.Pellicle and matrix, 2.Chromonemata, 3.Chromomeres, 4,Centromere, 5.Secondary

constrictions, 6.Satelite bodies.

1.Pellicle and matrix: Each chromosome is bounded by a membrane called pellicle. It is a very

thin and is formed of achromatic substance. This membrane encloses a jelly-like substance called

matrix. Chromonema is present with in matrix. The matrix is also formed of achromatic or

nongenetic material. The matrix forms the main bulk of the chromosome. It helps in keeping the

chromonemata within the bounds.

2.Chromonemata:With in the matrix of each chromosome contains two identical spirally coiled

filaments. They are called chromonemata. The nature and degree of coiling of chromonemata is

variable in meotic and mitotic chromosomes. In meotic chromosomes two distinct coils are

observed. One is called major coil, which consists of 10 to 30 gyres. The other is called minor

coil, which has more number of gyres. In mitotic chromosomes, a kind of coil similar to the

major coil is described. It is called somatic or standard coil. The coiling may be either paranemic

where the coil can be easily separated or plectonemic, where the coiling cannot be easily

separated.

3.Chromomere- In the meotic and the mitotic prophase, the chromonema shows alternate thik

and thin regions. The thich regions are bead-like structures and are called chromomeres. The

regions in between them are called interchromomeres. It is presumed that genes are located on

chromomeres. The chromomeres are structurally different from the remainder of the

chromonemata because of its ability to synthesize or accumulate the stainable nucleic acid or

nucleoprotrein.

4.Centromere or primary constriction-

It is a lighter staining narrow region. This narrow region is in the form of constriction. Hence it is

called primary constriction. The parts of chromosome which lie on the center side of the

chromosome are called arms. It is the site of kinetochore assembly.

Functions of centromere: 1.Spindel fibers are attached to centromer. 2. It helps for the

formation of spindle fibers. 3.It gives shape to the chromosome.

5.Secondary constriction: Occasionally, chromosomes contain additional constrictions other

than primary constriction. These are called secondary constrictions. There is absence of angular

deviation of the chromosomal segment. These constrictions are often associated with the

formation of nucleolus. Hence these are also known as nucleolar organizers. The chromosomes

with these structures are known as nucleolar chromosomes.

6.Satellite bodies: The small piece of chromosome located beyond the secondary constriction is

called satellite. It is round and elongated body. Chromosomes with satellite are called SAT

chromosomes. The satellites are usually single. But in some cases there may be two or more.

Chromosomes of prokaryotic cells: The prokaryotes-bacteria have single circular

chromosomes. The prokaryote chromosome base pairs range from 160,000 to 12,200,000. The

genes in prokaryotes are often organized in operons and do not contain introns. Operon is a

functional unit of genomic DNA.Prokaryotes do not possess nuclei. Instead, their DNA is

organized into a structure called the nucleoid. The nucleoid is a distinct structure present in a

bacterial cell. Prokaryotic chromosomes and plasmids are like eukaryotic DNA, generally

supercoiled.

Prokaryotic chromosome

Chemical composition of chromosomes: The chromosome is made up of 3 types of chemicals

namely proteins, DNA and RNA. (proteins-60%, DNA 35% and RNA 5%). The proteins present

in the chromosomes are called chromosomal proteins.These proteins helically wrap around

DNA. These proteins are basic in nature. They are of two types, namely protamines and histones.

Protamines: These chromosomal proteins are rich in the basic amino acid arginine. They are

found in spermatozoa of fish and snails.

Histones: These basic proteins are rich in arginine and lysine. There are 5 classes of histones,

namely H1, H2a, H2b, H3 and H5. The double helix of DNA wrap around the histone to form a

bead like structure called nucleosome. This DNA is the linker DNA. Each nucleosome consists

of 8 histones. It appears as a string of beads on a DNA strand. The core DNA makes 1.8 turn

around the nucleosome. Histones are not found in chromosomes of prokaryotic cells. In

eukaryotes histones serves to depress genes. They mask DNA.

Functions of chromosomes: 1.They controls the heredity.2. Control the metabolism of an

organism. 3. The heterochromatin helps in the formation of nucleolus.

Structure of chromatin: Within the cell nucleus DNA is not free but it is complexed with

proteins in a structure called chromatin. Chromatin consists of double –stranded DNA to which

large amounts of proteins and small amount of RNA are bound. There are three levels of

chromatin organization. a.Euchromatin, b. Heterochromatin, c. Metaphase chromosome.

a.Euchromatin: It is a lightly packed and stains less intensely. It is consists of DNA, RNA and

protein, that is rich in gene concentration. It is found in both prokaryote and eukaryotic cells.

b.Heterochromatin: It is the darkly stained condensed regions of chromosomes of the

interphase nucleus. It stains intensely, indicating tighter packing. This type found in satellite

sequences, centromere and telomere regions. When compared to euchromatin, heterochromatin

is late replicating. The DNA of heterochromatin is genetically inert and does not transcribe

mRNA for protein synthesis. In human, one X chromosome is totally heterochromatic and this

chromosome is called sex chromatin of Barr body. The heterochromatin regions contain genes of

rRNA, 5S RNA and tRNA. The DNA present in the heterochromatin is different from that of

euchromatin and is called satellite DNA or repetitive DNA. It is composed of short repeated

polynucleotide sequences.

Functions of chromatin: The primary functions of chromatin are: to package DNA into a smaller

volume to fit in the cell, to strengthen the DNA tp allow mitosis and meiosis and prevent DNA

damage, and to control gene expression and DNA replication. The primary components of

chromatin are histones that compact the DNA. Chromatin is only found in eukaryotic cells.

Prokaryotic cells have a different organization of their DNA which is referred to as a genophore

(a chromosome without chromatin)

.Genome complexity: (to be covered later)

e) Cell signaling: Communication between cells and their environment, ion channels, signal

transduction pathways (MAP kinase, P38 kinase, JNK, Ras and P13-kinase pathways, and

biosensors.

Signal transduction occurs when an extracellular signaling molecule activates a cell surface

receptor. In turn, this receptor alters intracellular molecules creating a response. There are two

stages in this process.

1.A signaling molecule activates a specific receptor on the cell membrane.

2.A second messenger transmits the signal into the cell, eliciting a physiological response.

Signal transduction through receptors: Receptors can be roughly divided into two major classes:

intracellular receptors and extracellular receptors.

Extracellular receptors: These receptors are integral transmembrane proteins. They span the

plasma membrane of the cell, one part of the receptor on the outside of the cell and the other on

the inside. Signal transduction occurs as a result of a ligand binding to the outside, the molecule

does not pass through the membrane. This binding stimulates a series of events inside the cell.

Upon binding, the ligand induces a change in the conformation of the inside part of the receptor.

These result in either the activation of an enzyme in the receptor or the exposure of a binding site

for other intracellular signaling proteins within the cell, eventually propagating the signal

through the cytoplasm.

Ionotropic receptors –

These are transmembrane ion channels. Many cellular functions require the passage of ions and

other hydrophilic molecules across the plasma membrane. Ionotropic receptors regulate these

processes.

Main functions of ion channels

Neurotransmission, cardiac conduction and muscle contraction.

Types of ion channel receptors-

1. Voltage gated ion channels-2. Ligand gated ion channels, 3. G-Protein gated ion channels

1. Voltage gated ion channels- Voltage-gated ion channels are a class of transmembrane ion

channel that are activated by changes in electrical potential difference near the channel. These

types of ion channels are common in many types of cells including neurons..

a.Voltage-gated sodium channels: Sodium channels consist of a large α subunit that associates

with other proteins, such as β subunits. Alpha subunit forms the core of the channel. The α-

subunit has four repeat domains, labeled I through IV, each containing six membrane-spanning

regions, labeled S1 to S6. The S4 region acts as the channel's voltage sensor. When the cell

membrane is at its resting potential, the activation gates of the voltage gated sodium channels are

closed and the inactivation gates are open. Voltage gated potassium channels are closed.

Depolarization is initiated by a stimulus which makes the membrane potential more positive,

causing the voltage gated sodium channels to starts to open. As threshold is reached many

sodium channels open. Sodium ions diffuse across the membrane causing depolarization.

Voltage gated potassium ion channels also begin to open, but more slowly. Therefore,

depolarization occurs because more sodium ions diffuse in to the cell than K+ diffuse out of it.

As the membrane potential approaches maximum depolarization, the inactivation gates of the

channel begin to close and diffusion of sodium ions decreases. Then the activation gate gets

closed and inactivation gate gets opened. The potassium channels remain open and potassium

ions continue to diffuse out of the cell. After the voltage gated potassium ion channel close, the

resting membrane potential is restored. E.g – Expressed on the nerve axons. These are important

for the conduction of the nerve impulse and for the initiation of the action potential.

b.Voltage-gated calcium channels: These channels consists of α1,α2,δ, β, and γ subunits. These

channels play an important role in both muscle contraction and neuronal excitation, the release of

neurotransmitters. These channels are found in excitable cells (e.g., muscle, neurons,etc) with

permeability to the ion Ca2+

.

Types-.

a. L-Long lasting-slow. These are gated by high voltage. These are expressed on the skeletal

muscles, heart muscles, osteoblasts, etc. Functions- SMC, cardiac muscle contraction.

b.P-/ Q type- These are gated by high voltage type and are expressed on the purkinje neurons of

the cerebellum. Q type expressed on the cerebellar granule cells. Function- Release of NTs.

c.N(neuronal)-type- These are also gated by high voltage. These are expressed on throughout

the brain. Function- Release of NTs.

d. T- Transient- These are gated by low voltage. These are expressed on SAN, AVN (pace

maker cells), neurons and osteocytes. Function- Sinus rhythm.

c.Voltage-gated potassium channels (KV): These are transmembrane channels specific for

potassium and sensitive to voltage changes in the cell’s membrane potential, they play a crucial

role in returning the depolarized cell to a resting state. The typical voltage-gated K. channel is

an assembly of four identical transmembrane subunits surrounding a central pore. Each subunit

has six transmembrane crossings (S1–S6), with both N- and C termini on the intracellular side of

the membrane. The narrowest part of the pore is formed between S5-S6 loops; it is selectively

permeable to the potassium ions. The S4 consists of positive charges and it is considered as

voltage sensor.

.

Voltage gated potassium channel.

d.Voltage gated proton channels: If there is accumulation of electrons due to depolarization

within the cell, the proton channels get open, hence the protons (H+) leaves the cell through the

voltage gated proton channels. When bacteria or other microbes are engulfed by phagocytes

(e.g.eosinophils, neutrophils, and macrophages), the enzyme NADPH oxidase assembles in the

membrane and begins to produce reactive oxygen species (ROS) that help to kill the bacteria.

NADPH oxidase is electrogenic, moving electrons across the membrane, and proton channels

open to allow proton flux to balance the electron movement electrically.

2. Ligand gated ion channels (LGICs): These are a group of transmembrane ion channels that

are opened or closed in response to the binding of a chemical messenger (i.e.ligand), such as a

neurotransmitter. The binding site of endogenous ligands on LGICs is normally located on an

allosteric binding site . The ligand binding causes opening or closing ot the ion channel. The ion

channel is usually selective to one or more ions like Na+, K

+, Ca

2+ or Cl

-. Example -acetylcholine

nicotinic receptors (NN).

Structure-It consists of 4-5 protein subunits.(ααβγδ). Each subunit contains 4 transmembrane

polypeptides (M1, M2, M3, and M4). (M1-M2), (M2-M3)- have short connecting loops. (M3-

M4)-have long loop with phosphorylation site by different second messenger proteins. There is

extracellular N terminal and intracellular C terminal which is short. Adjacent M2 of all the

protein subunits form the ion pore.

Types- Mainly 3 types

I.Cys-loop receptors: They have pentameric structure. They contain a characteristic loop

formed by a disulfide bond between two cysteine residues

a.Anionic Cys - loop receptors- Eg GABAA and glycine receptor (GlyR).

b.Cationic Cys-loop receptors- Eg-5HT3 receptor, nAchR, Zinc activated ion channel (ZAC).

II.Iononotropic glutamate receptors- They bind with the NT glutamate. They have tetrameric

structure. Eg.AMPA (alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid).Kainate and

NMDA (N-Methyl-D-aspartate receptors) receptors.

III. ATP gated ion channel receptors: The nucleotide ATP acts as NT. These receptors are

activated when these NTs bind with the binding sites. They have trimeric structure. Eg- PTX

(purinoceptor).

Voltage gated ion channel

G-Protein gated ion channels- These ion channels are gated by G proteins (guanine nucleotide-

binding proteins).

Types of G-Protein gated ion channels –

a.G.Protein gated potassium channels

b.G.Protein gated calcium channels

c.G.Protein gated sodium channels

d.G.Protein gated chloride channels

a.G.Protein gated potassium channels- Locations-Eg Atrial muscle, SAN of the heart- When

vagus nerve is stimulated, Ach is secreted, this binds with M2 receptors. The Gβγ subunit binds

with K+

channel. This leads to the removal of potassium from the cell causing hyperpolarization.

This controls the HR.

G.Protein gated potassium channels also located in the brain. GABAB activation by GABA-

>activation of K+ channel-> hyperpolarization.

b.G.Protein gated calcium channels-These are present on the neuronal membranes and the

heart. The activation of the G Proteins via effectors AC, PLC and second messengers- cAMP, IP3

and DAG causes the opening of the Ca2+

channels by protein phosphorylation.

c.G.Protein gated sodium channels- They are in investigational stage.

d. G.Protein gated chloride channels- They are in investigational stage.

G protein-coupled receptors (GPCRs) - These are 7 transmembrane domain receptors. These

are also called as serpentine receptors, as they look like snake. Structurally GPCRs are

characterized by an extracellular N-terminus, followed by seven transmembrane (7-TM) α-

helices (TM-1 to TM-7) connected by three intracellular (IL-1 to IL-3) and three extracellular

loops (EL-1 to EL-3), and finally an intracellular C-terminus. Ligand- It is an ion or a molecule,

or a molecular group that binds to another chemical entity to form a larger complex.

Ligands of the GPCRs- Some examples- Adenosine, bradykinin, γ-aminobutyric acid

(GABA),peptides, opsins, somatostatin, vasopressin, biogenic amines (e.g., dopamine,

adrenaline, noradrenaline, histamine, glucagon, acetylcholine, and serotonine) lipid derived

autocoids (e.g., prostaglandins, PAF, and leucotrienes) and peptide hormones (e.g., calcitonin,

follicle-stimulating hormone(FSH), gonadotropin-releasing hormone (GnRH), neurokinin,

thyrotropin-releasing hormone TRH and oxytocin). If ligand is not identified for a GPCR, then it

is called as orphan receptor. If a ligand for an orphan receptor is later discovered, such receptor

is known as adopted orphan receptor.

When a ligand activates the G-protein coupled receptor, it induces a conformational change in

the receptor that allows the receptor to function as a guanine nucleotide exchange factor (GEF)

that exchanges GDP for GTP on the Gα subunit. This exchange triggers the dissociation of the

Gα subunit bound to GTP, from the Gβγ dimer and the receptor. Both Gα-GTP and Gβγ can then

activate different signaling cascades (or second messenger pathways) and effector proteins. The

Gα subunit will hydrolyze the attached GTP to GDP by its inherent enzymatic activity, allowing

it to re-associate with Gβγ and starting a new cycle.

G proteins (guanine nucleotide-binding proteins)-These are membrane associated heterotrimeric

proteins. They are also known as large proteins. These proteins are activated by G protein-

coupled receptors. These proteins are made up of α,β and γ subunits.

Alpha subunit (Gα)- This subunit consists of two domain, the GTPase domain and the alpha-

helical domain. There are about 20 different Gα subunits. They are separated into 4 main

families.

1. Gi family-

Gi/o Alpha subunits- αi and αo - Signal transduction- inhibition of adenylate cyclase, opens K+

channels (via β/γ subunits), closes Ca2+

channels.

Examples- Muscarinic M2 and M4 receptors, α2 adrenoreceptors, 5- HT1receptors, H1 and H2

receptors, D2 receptors.

Gt - Alpha subunit- αt – Signal transduction-activation of phosphodiesterase. Example-

Rhodopsin receptor- vision.

Ggust- Alpha subunit- αgust. Signal transduction- activation of phosphodiesterase. Example-

Taste receptors- taste.

2.Gs family- Gs- Alpha subunit-αs. Signal transduction- activation of adenylate cyclase.

Examples- beta adrenergic receptors, 5HT4, 5HT6 and 5HT7 receptors. D1 receptors, H2

receptors. Golf- Alpha subunit-Golf- Signal transduction- activation of AC. Examples- olfactory

receptors- smell.

3.Gq family-Alpha subunits- αq, α11,α14,α15α16. Signal transduction – activation of phospholipase

C (PLC). Examples- α1 receptors, H1 receptor, 5HT2 receptors, M1, M3 and M5 receptors.

4.G12/13 family- Alpha subunits-α12, α13. Signal transduction- activation Rho GTPases

Adenylate cyclase (AC) - Adenylyl cyclase is the enzyme that synthesizes cyclic adenosine

monophosphate or cyclic AMP from adenosine triphosphate (ATP). Cyclic AMP functions as a

“second messenger” to relay extracellular signals to intracellular effectors, particularly protein

kinase A.

Structure and Function

Adenylyl cyclases are integral membrane

proteins that consist of two bundles of six

transmembrane segments. Two catalytic

domains extend as loops into the cytoplasm.

The activated adenylyl cyclase catalyses the

conversion of ATP to cyclic AMP, this leads to an increase in intracellular levels of cyclic AMP.

There are at least nine isoforms of adenylyl cyclase.

Regulation of activity- Binding of a stimulatory G alpha (Gs) enhanced activity while binding of

an inhibitory G alpha (Gi) inhibited cyclase activity.

cAMP- Cyclic AMP is an important second messenger. It forms, as shown, when the membrane

enzyme adenylyl cyclase is activated (as indicated, by the alpha subunit of a G protein).

The cyclic AMP then activates specific proteins. Some ion channels are gated by cyclic AMP.

But an especially important protein activated by cyclic AMP is protein kinase A. The activated

PKC phosphorylate certain cellular proteins. The scheme below shows how cyclic AMP

activates protein kinase A.

Functions- cAMP is a second messenger, used for intracellular signal transduction, such as

transferring the effects of hormones like glucagon and adrenaline, which cannot pass through the

cell membrane. It is involved in the activation of protein kinases and regulates the effects of

adrenaline and glucagon. It also regulates the passage of Ca2+

through ion channels.

Degradation of cAMP-

Phospholipase C (PLC)- PLC cleaves a phospholipid present in the cell membrane.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is cleaved into diacyl glycerol (DAG) and inositol

1,4,5-triphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble

structure into the cytosol. IP3 then diffuses through the cytosol to bind to second messenger gated

calcium channels in the endoplasmic reticulum (ER). This increases the cytosolic concentration

of calcium. The calcium is responsible for the cellular activity. The membrane bound DAG

activate the protein kinase C(PKC). The activated PKC phophorylate the other molecules,

leading altered cellular activity.

Protein kinase C(PKC)- is a family ofenzymes that are involved in controlling the function of

other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino

acid residues. PKC enzymes in turn are activated by signals such as increases in the

concentration of DAG or Ca2+

. Hence PKC enzymes play important roles in several signal

transduction cascades.

Examples-Some protein kinases are activated by Ca++

-calmodulin. Protein Kinase A is activated

by cyclic-AMP (cAMP).

Guanylate cyclase C- (GC-C) Guanylate cyclase catalyzes the reaction of guanosine

triphosphate (GTP) to 3’,5’- cyclic guanosine monophosphate (cGMP) and pyrophosphate.

Protein Kinase

O

ProteinOH + ATP ProteinOPO + ADP

O Pi H2O Protein Phosphatase

H3N+

C COO

CH2

OH

H

serine (Ser)

H3N+

C COO

CH OH

CH3

H

threonine (Thr)

cGMP serves as second messenger for the neurotransmitters atrial natriuretic peptide (ANP) and

NO.

Second messenger systems

Second messengers are molecules that relay signals from receptors on the cell surface to target

molecules inside the cell, in the cytoplasm or nucleus. They relay the signals of hormones like

adrenaline, histamine, serotonin, growth factors, and others, and cause change in the cellular

activity. They amplify the strength of the signal. Secondary messengers are a component of

signal transduction cascades.

Types- There is three basic types of secondary messenger molecules:

1.Hydrophobic molecules: Water-insoluble molecules, like diacylglycerol (DAG), and

phosphatidylinositols. These are located near cell membrane.

2. Hydrophilic molecules: Water-soluble molecules, like cAMP, cGMP, IP3, and Ca2+

, that are

located within the cytosol.

3.Gases: NO, CO and H2S which can diffuse both through cytosol and across cellular

membrane.

Hydrophobic molecules: Diacylglycerol (DAG)- It is a glyceride consisting of two fattyacid

chains covalently bonded to a glycerol molecule through ester linkages. Diacylglycerols ( or

diglycerides) are esters of the trihydric alcohol glycerol in which two of the hydroxyl groups are

esterified with long-chain fatty acids. They can exist in three stereochemical forms.

When neurotransmitters bind with GPCRs activate the intracellular enzyme PLC. The activated

PLC phospholipids-specially phosphatidylinositol-4,5-bisphosphate (PIP2) which is found in the

inner layer of the plasma membrane. Hydrolysis of PIP2 yields two products: DAG and IP3.

DAG-It remains in the inner membrane of the cell and bind with PKC, PKC is activated in

presence of Ca2+

ions. The other second messenger IP3 bind with SR, to release Ca2+

ions. The

released Ca2+

binds with PKC to activate it. Hence both DAG and Ca2+

are required for the

activation of PKC.

Phosphatidylinositols: Phosphatidylinositol 4,5-bisphosphate-(PIP2): It is a minor

phospholipid component of cell membrane. The most common fatty acids present in PIP2 are

stearic acid at position 1 and arachidonic acid at position 2. If a ligand like acetylcholine binds

with GPCR, activates the αq. The αqGTP sub unit of G protein activates the membrane bound

effector PLC. PIP2 is a substrate for hydrolysis by PLC. The products of this reaction are IP3 and

DAG. DAG remains on the cell membrane and in presence of Ca2+

activates PKC. The activated

PKC phosphorylate other cytosolic proteins. The phosphorylated cellular proteins are responsible

for altered cellular activity. The IP3 enters the cytoplasm and activates the IP3 receptors present

on the sarcoplasmic or enoplasmic reticulum cell membrane, which opens the Ca2+

channels on

the membrane. This triggers the Ca2+

concentration within the cytoplasm. The triggered Ca2+

ions are responsible for the altered cellular activity.

2. Hydrophillic molecules: Water soluble molecules, like cAMP, cGMP, IP3 and Ca2+

that are

located within the cytosol..

3. Gases: Nitric oxide (NO), CO, H2S. Which can diffuse both through cytosol and across

cellular membranes.-

Nitric oxide is a highly reactive gas. In the body cells NO is formed by an enzyme NO synthases

(NOS). These are 3 types of NOS.

1.nNOS (or NOS-1):Found in neurons.

2.eNOS (or NOS-3): Found in the endothelium of the blood vessels.

3.iNOS(or NOS-2):Found in the macrophages.

Synthesis of NO: NOS produce NO from arginine with the aid of molecular oxygen and

NADPH; NO diffuses freely across the cell membrane.

The synthesized NO from the endothelial cells then diffuses into the surrounding smooth muscle

fibre. Within the smooth muscle cells, NO activate the soluble guanylyl cyclase. It is only the

known receptor for the NO. The activated soluble guanylyl cyclase produces another second

messenger-cGMP. An increase in cGMP and the subsequent activation of protein kinase G. PKG

has lead to relaxation of smooth muscle, including phosphorylation of the K+

channel to produce

a hyperpolarization of the smooth muscle, a decrease in Ca2+

flux, and an activation of myosin

light chain (MLC) phosphatase. It inhibits smooth muscle contraction by dephosphorylating

myosin light-chain fibers. It counters the MLCK, which promotes contraction by

phosphorylating myosin light-chain proteins. Hence NO causes smooth muscle relaxation.

Carbon monoxide: It is a colorless, odorless, and tasteless gas which is slightly lighter than air.

It is highly toxic to humans and animals in higher quantities, although it is also produced in

normal metabolism in low quantities, and is thought to have some normal biological functions.

Carbon monoxide consists of one carbon atom and one oxygen atom, connected by a triple bond

which consists of two covalent bonds. It is generated as a by-product of heme breakdown

catalyzed by heme oxygenase. It is generated as a by-product of heme breakdown catalyzed by

heme oxygenase. CO also stimulates the formation of cGMP. The MOA of CO is similar to NO.

Hydrogen sulphide (H2S): H2S is formed endogenously from L-cysteine or L-methionine by

two enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), and

normally circulates in blood. H2S dilates the blood vessel by stimulating the K+ channels in the

vascular smooth muscles. The loss of potassium ions causes hyperpolarization and this relaxes

the smooth muscles.

e) Cell signaling: Communication between cells and their environment, ion channels, signal

transduction pathways (MAP kinase, P38 kinase, JNK, Ras and P13-kinase pathways, and

biosensors.

MAP kinase: Mitogen-activated protein (MAP) kinases are serine/threonine- specific protein

kinases that respond to extracellular stimuli (mitogens, osmotic stress, heat shock and pro-

inflammatory cytokins) and modulate the cellular activities, such as proliferation, gene

expression, differentiation, mitosis, cell survival, and apoptosis. A serine/threoninie protein

kinase is a kinase enzyme that phosphorylates the OH group of serine or threonine (which have

similar side chain). At least 125 of the 500 + human protein kinases are serine/threonine kinases.

MAPK signal transduction pathway: MAPK pathway is a chain of proteins in the cell that

communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the

cell. The signal starts when a growth factor binds to the receptor on the cell surface and ends

when the DNA in the nucleus expresses a protein and produces some change in the cell, such as

cell division. This pathway includes many proteins, including MAPK, which communicated by

adding phosphate groups to a neighboring proteins, which acts as an “on” or “off” switch. When

one of the proteins in the pathway is mutated, it can be stuck in the “on” or “off” position, which

is a necessary step in the development of many cancer cells. Drugs that reverse the “on” or “off”

switch are being investigated as cancer treatments.

Receptor-linked tyrosine kinases such as epidermal growth factor receptor (EGFR) are activated

by extracellular ligands. Binding of the epidermal growth factor (EGF) to the EGFR activates the

tyrosine kinase activity of the cytoplasmic domain of the receptor. The EGFR becomes

phophorylated on tyrosine residues. Docking (attacking) proteins such as GRB2 (growth factor

receptor bound protein) contains an SH2 domain that binds to the phosphotyrosine residues of the

activated receptor. GRB2 binds to the guanine nucleotide exchange factor SOS(Son of sevenless).

This activates SOS. The activated SOS promotes the removal of GDP from Ras. Ras can then

bind GTP and become active. Apart from EGFR, other cell surface receptors that activate this

pathway via GRB2 include Fibroblast growth factor receptor (FGFR) and PDGFR (platelet

derived growth factor receptor).

Activated Ras activates the protein kinase activity of RAF kinase (A-RAF,B-RAF and C-RAF).

RAF kinase phophorylates and activates MEK (MEK1 and MEK2). It is also called MAPKK.

MEK phosphrylates and activates a mitogen-activated protein kinase (MAPK). RAF, MEK and

MAPK are all serine/threonine – selective protein kinases. In technical sense, RAF, MEK and

MAPK are all mitogen-activated kinases. MAPK was originally called “extracellular signal-

regulated protein kinases-ERK). Three of the many proteins phosphrylated by MAPK are RSK,

MNK and MYC. The activated RSK phosphrylates ribosomal protein S6. Activated MNK

regulates the activities of several transcription factors through the phophorylation of CREB

(cAMP response element –binding) and The phosphorylated MYC (a gene) also regulates several

transcription factors.

Clinical significance of MAPK transduction pathway: Uncontrolled growth is a necessary

step for the development of all cancers. In many cancers (e.g.melanoma), a defect in the MAPK

pathway leads to that uncontrolled growth. Many compounds can inhibit in the MAPK pathway,

and therefore are potential drugs for treating cancers.e.g. Hodgkin disease.

Some of the anticancer drugs act by inhibiting MAPK pathway are-

a. Sorafenib, vemurafenib - RAF kinase inhibitors.

b.Selumetinib- a MAPK inhibitor.

p38MAPK transduction pathway. The family of MAPK is composed of three major groups:

the extracellular regulated kinases (ERKs), the C-Jun N-terminal kinases (JNKs) and the p38

MAPKs. The p38 MAPK group consists of four members: p38α, p38β, p38γ and p38δ. These are

activated by a variety of environmental stresses (such as UV irradiation, heat shock,etc) and

inflammatory cytokines. Stress signals are delivered to this cascade by members of small

GTPases of the Rho family (Rac, Rho, Cdc42). The Rho family is a family of small signaling G

protein (a GTPase), and is a subfamily of the Ras superfamily. Rho proteins have been described

as “ molecular swithes” and play a role in cell proliferation, apoptosis, gene expression and other

common cellular function. In the other cascade MAPKKK phosphorylates and activates

MAPKK, which in turn phosphorylates and activates MAPK. This cascade is initiated by a signal

coming from outside the cell.

The activated p38MAPK is involved in regulation of Hsp 27 and MAPKAPK-2 (MAPK

activated protein kinase) and several transcription factors including ATF2, MEF-2, ELK-1 and

MSK1. Hsp 27 is an ATP –independent chaperone that protect the cell against apoptosis through

various mechanisms.

The role of p38 MAPK in the G2/M cell cycle checkpoint: Exposure to ultraviolet (UV), γ

irradiation and chemotherapeutic drugs such as etoposide results in the generation of DNA

double strand breaks (DSBs). In response to DSBs, p38 MAPK is activated and leads to the

establishment of a G2/M cell cycle checkpoint. These proteins enforce a G2/M checkpoint by

either directly or indirectly inactivating CDK2/cyclin B complex (as this complex is essential for

the progression of the cell cycle from G2 to M.). p38 MAPK can also induce a G2/M checkpoint

through the phosphorylation and inhibition of the phosphatase.

The role of p38 MAPK in the G1/S cell cycle checkpoint: In response to stimuli such as

osmotic stress, ROS activates p38 MAPK. The activated p38 MAPK induces G1/S cell cycle

checkpoint by the activation of the inhibitory protein p-53. This leads to the accumulation of

another inhibitory protein p-21. This inhibitory protein inhibits the CDK2 results in G1/S

restriction.

JNK transduction pathway: (c-Jun-N terminal kinases) JNKs belongs to second group of

MAPKs. These kinases bind and phosphorylate c-Jun on ser-63 and ser-73 within its

transcriptional activation domain. These kinases are responsive to stress stimuli, such as

cytokines, ultraviolet irradiation, heat shock, osmotic shock. They also play a role in T cell

differentiation and the cellular apoptosis pathway. Activation of JNKm is carried by two MAP

kinases, MKK4 (MAP kinase kinase4) and MKK7 (MAP kinase kinase7) and JNK can be

inactivated by Ser/Thr and Tyr phophatases. There are three isoforms of JNK- JNK 1 and JNK 2

are found in all cells and tissues. JNK3 is found mainly in brain and also in the heart and testes.

Inflammatory signals, changes in levels of reactive oxygen species, ultraviolet radiation, protein

synthesis inhibitors, and a variety of stress can activate JNK. JNK, by phosphorylation, modifies

the activity of numerous proteins that reside at the mitochondria or act in the nucleus. By

activating or inhibiting the other molecules, JNK regulates several important cellular functions

including cell growth, differentiation, survival and apoptosis.

The molecules that are activated by JNK include - c-Jun, ATF2, ELK1, SMAD4, p53 and HSF1.

The molecules that are inhibited by JNK include- NFAT4, NFATC1 and STAT3.

cJun- It is a protein and it is activated through double phosphorylation by JNK. The activated

cJun combines with c-Fos (onco-gene) and forms AP-1 (activator protein-1) – a transcription

factor.

ATF2- (activation transcription factor-2)- This protein is stimulated by JNK. It is involved in the

growth and progression of mammalian skin tumors.

ELK1: (E-twenty six ETS-like transcription factor) This protein is stimulated by JNK. It is also

one of the transcription factors.

SMAD4 – These signal transduction proteins are activated and phosphorylated by JNK. The

activated SMAD4 molecules move to the cell nucleus. In the nucleus, the SMAD4 proteins bind

to specific areas of DNA where it controls the activity of particular genes and regulates cell

growth and division (proliferation).

P53- It is known as protein53 or tumor protein53. It is a tumor suppressor (anti-oncogene)

protein. It regulates the cell cycle and function as a tumor suppressor that is involved in

preventing cancer. It has been described as “ the guardian of the genome” as it prevents the

genome mutation. JNK activate the p53 by the phosphorylation.

HSF1: Heat shock protein 1 is a protein. It is exist in an inactive form by binding with

Hsp40/Hsp70 and Hsp90. Upon stress, such as elevated temperature, it is activated by JNK. The

activated HSF1 is then transported into the nucleus where it is hyperphosphorylated and binds to

DNA containing heat shock elements. This result in growth arrest or apoptosis.

The molecules that are inhibited by JNK include- NFAT4, NFATC1 and STAT3

NFAT4: It is a transcription factor that plays a role in the regulation of gene expression in T cells

and immature thymocytes. This factor is inhibited by JNK.

NFATC1: Nuclear factor of activated T-cells, cytoplasmic 1 is a protein. This transcription

factor is inhibited by JNK. These transcription factors play a role in inducible gene transcription

during immune response. This is the molecular target for the immunosuppressive drugs such as

cyclosporine A.

STAT3 (Signal transducer and activator of transcription 3) It is a transcription factor.