In the last article, we have studied monosaccharides. In this article, we shall study disaccharides and polysaccharides.

Disaccharides:

Di-saccharides on hydrolysis give two molecules of monosaccharide. They on hydrolysis with dilute acids or enzymes yield two molecules of either the same or different monosaccharides.

e.g. Cane sugar (Sucrose) (C₁₂H₂₂O₁₁)on hydrolysis gives one molecule of glucose and one molecule of fructose, Maltose (C₁₂H₂₂O₁₁) on hydrolysis gives two molecules of glucose, Lactose (C₁₂H₂₂O₁₁) on hydrolysis gives one molecule of glucose and one molecule of galactose.

• Disaccharides are crystalline, water-soluble, and sweet in taste.
• They have the general formula (C₁₂H₂₂O₁₁).
• The two monosaccharides are joined together by an oxide linkage formed by the loss of a water molecule. Such a linkage between two monosaccharide units through oxygen atom is called glycosidic linkage.

Examples of Disaccharides:

Sucrose:

One of the common disaccharides is sucrose which on hydrolysis gives an equimolar mixture of α -D-Glucapyranose and β-D-Fructofuranose. These two monosaccharides are held together by a glycosidic linkage between C1 of α-glucose and C2 of β-fructose.

Since the reducing groups of glucose and fructose are involved in the glycosidic bond formation, sucrose is a non-reducing sugar.

Sucrose is dextrorotatory but after hydrolysis gives dextrorotatory glucose and laevorotatory fructose. Since the laevorotation of fructose (–92.4°) is more than dextrorotation of glucose (+ 52.5°), the mixture is laevorotatory. Thus, hydrolysis of sucrose brings about a change in the sign of rotation, from dextro (+) to laevo in the sign of rotation, from Dextro (+) to Laevo (–) and the product is named as invert sugar.

Maltose:

Another disaccharides, maltose is composed of two α-D-glucose units in which C1 of one glucose (I) is linked to C4 of another glucose unit (II).

The free aldehyde group can be produced at C1 of second glucose in solution and it shows  reducing properties so it is a reducing sugar

Cellobiose:

Cellobiose is obtained by partial hydrolysis of cellulose, C1 of one β-D-Glucapyranose is linked to C4 of another β-D-Glucapyranose by glucosidic linkage. Thus cellobiose contains 1→ 4 β- glucosic bond.

Cellobiose is reducing sugar because free aldehyde group can be produced at C1 in second glucose molecule

Lactose:

It is more commonly known as milk sugar since this disaccharide is found in milk. It is composed of β-D-galactose (β-D-Galactopyranose) and β-D-glucose (β-D-Glucopyranose). The glucosidic linkage is between C1 of β-D-galactose (b-D-Galactopyranose) and C4 of β-D-glucose (β-D-Glucopyranose). Hence it is also a reducing sugar.

Reducing Sugars:

The saccharides in which aldehydic and ketonic groups are free are called reducing sugars. They reduce Fehling’s solution and Tollen’s reagent. They contain either α-hydroxy aldehyde or α -hydroxy ketone group or contain cyclic hemiacetal or cyclic hemiketal structures.

All monosaccharides are reducing sugars. Diasaccharides like maltose, lactose and cellobiose are reducing sugars.

Nonreducing sugars:

The saccharides in which aldehydic and ketonic groups are not free are called non-reducing sugars. They do not reduce Fehling’s solution and Tollen’s reagent. They contain stable acetal or ketal structures which cannot be opened into the free carbonyl group.

Sucrose, Starch, Cellulose, Glycogen, Dextrin are reducing sugars.

Hemiacetal and Hemiketal Structures:

They are formed when an alcohol oxygen atom adds to the carbonyl carbon of an aldehyde or a ketone. When alcohol adds to an aldehyde, the result is called a hemiacetal; when alcohol adds to a ketone the resulting product is a hemiketal.

This happens through the nucleophilic attack of the hydroxyl group at the electrophilic carbonyl group. Since alcohols are weak nucleophiles, the attack on the carbonyl carbon is usually promoted by protonation of the carbonyl oxygen.

PolySaccharides:

Carbohydrates which on hydrolysis give indefinite or large no. of monosaccharides (more than 10)  are called polysaccharides. They contain a large number of monosaccharide units joined together by glycosidic linkages. These are the most commonly encountered carbohydrates in nature. They mainly act as food storage or structural materials.

• They are natural polymeric carbohydrates. They are insoluble in water, amorphous, and tasteless.
• They are nonsugars.
• They have the general formula (C6H10O5)n.  e.g. Starch, Cellulose, Inulin, Dextrin, etc
• Cellulose is a linear polymer of β-Glucose units while starch is a branched polymer of a-glucose units.

Examples of Polysaccharides:

Starch:

Starch is the main storage polysaccharide of plants. It is the most important dietary source for human beings. High content of starch is found in cereals, roots, tubers, and some vegetables. It is a polymer of α-glucose (α-D-Glucopyranose) and consists of two components Amylose and Amylopectin.

Amylose is a water-soluble component which constitutes about 15-20% of starch. Chemically amylose is a long unbranched chain with 200-1000 α-D-(+)-glucose units held by C1– C4 glycosidic linkage.

Amylopectin is insoluble in water and constitutes about 80-85% of starch. It is a branched chain polymer of α-D-glucose units in which chain is formed by C1–C4 glycosidic linkage whereas branching occurs by C1–C6 glycosidic linkage

Glycogen:

The carbohydrates are stored in the animal body as glycogen. It is also known as animal starch because its structure is similar to amylopectin and is rather more highly branched. It is present in the liver, muscles, and brain. When the body needs glucose, enzymes break the glycogen down to glucose (hydrolysis). Glycogen is also found in yeast and fungi.

Importance of Carbohydrates:

• Carbohydrates are essential for life in both plants and animals.
• They form a major portion of our food. Honey has been used for a long time as an instant source of energy by ‘Vaids’ in Ayurvedic system of medicine.
• Carbohydrates are used as storage molecules as starch in plants and glycogen in animals.
• The cell wall of bacteria and plants is made up of cellulose. We build furniture, etc. from cellulose in the form of wood and clothe ourselves with cellulose in the form of cotton fibre.
• They provide raw materials for many important industries like textiles, paper, lacquers and breweries.
• Two aldopentoses viz. D-ribose and 2-deoxy D-ribose (Section are present in nucleic acids. Carbohydrates are found in biosystem in the combination with many proteins and lipids.

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In the last article, we have seen what are carbohydrates and how are they classified. Based on hydrolysis behaviour the carbohydrates are classified into three types. a) Mono-Saccharides b) Oligo-Saccharides and c) Poly-Saccharides. In this article, we shall study monosaccharides in detail particularly glucose and fructose.

Monosaccharides:

Carbohydrates which are basic units or which cannot be hydrolyzed further are called monosaccharides.

Characteristics of mono-saccharides:

• Carbohydrates which are basic units or which cannot be hydrolysed further are called monosaccharides.
• They are basic units of carbohydrates.
• They cannot be hydrolysed further into small units.
• They contain six carbon atoms in a molecule.
• They are water-soluble and sweat in taste.
• Depending upon the presence of an aldehydic group or a ketonic group they are further subclassified into aldoses and ketoses respectively.

Aldoses: The monosaccharides containing the aldehydic group are called aldoses. Examples: Aldopentose C₅H₁₀O₅ – Arbaniose, Xylose, Ribose. Aldohexose C₆H₁₂O₆ – Glucose, Galactose

Ketoses: The monosaccharides containing the ketonic group are called ketoses. Examples: Ketopentose C₅H₁₀O₅ – Ribulose.Ketohexose C₆H₁₂O₆ – Fructose.

Preparation of Glucose:

From Sucrose (Cane sugar): (Laboratory Method):

When powdered cane sugar is heated with a concentrated alcoholic solution of HCl on a water bath for about 2 hours at about 323 K, glucose is formed.

It is insoluble in alcohol while fructose is soluble in alcohol. Hence glucose crystallizes out first leaving fructose in the solution.  A few crystals of glucose may be added to the solution for quicker crystallization. This is known as seeding. Purification is done by recrystallizing it from methanol.

From Starch: (Commercial Method):

Starch on hydrolysis with dilute sulphuric acid by heating under 3 to 5 atmospheric pressure gives glucose.

When hydrolysis is complete the excess of unreacted sulphuric acid is neutralised with calcium carbonate and filtered to remove the precipitate of calcium sulphate.

CaCO₃ + H₂SO₄   →   CaSO₄   + H₂O + CO₂ ­

The filtrate which contains glucose and one molecule of water is decolorized using animal charcoal. The clear solution is then evaporated in a vacuum to get a thick syrup on cooling crystallizes to give glucose-monohydrate. It is recrystallized from methanol.

Structure of Glucose:

Open Chain Structure:

Following chemical reactions of glucose confirm its open chain structure

• On prolonged heating with HI it gives n-hexane suggesting that all the six carbons are linked in a straight chain.

• Hydroxylamine condenses with an aldehydic group to form glucose-oxime.

• Hydrogen cyanide adds to an aldehydic group to form cyanohydrin.

• On oxidation by a mild oxidizing agent like bromine water, it gives gluconic acid, which shows that the carbonyl group is the aldehyde group.

• Glucose, as well as gluconic acid on oxidation by dilute nitric acid, gives dicarboxylic acid, saccharic acid, which shows the presence of the alcoholic group.

• On acetylation by acetic anhydride it gives glucose-pentaacetate., which confirms the presence of five hydroxyl group. As glucose is a stable compound these five hydroxyl group must be on five different carbon atoms.

Challenges to open chain structure or the need of cyclic structure:

The following points indicate the absence of a free aldehyde group in glucose.

• In spite of having an aldehyde group it does not give a condensation reaction with 2,4 dinitro-phenyl hydrazine
• Glucose-pentaacetate does not condense with hydroxylamine
• It is found to exist in two different crystalline forms α and β called anomers.

Haworth Projection of Glucose:

The cyclic structure in Haworth projection depicts the ring as being flat. The substituents that are to the right in a Fischer projection formula are down and those to the left are up in the corresponding Haworth projection formula. Orient the Haworth projection formula with the ring oxygen at the back and the anomeric carbon at the right.

For carbohydrates of D series a) If hydroxyl is down, the configuration of anomeric carbon is α and b) If hydroxyl is up, the configuration of anomeric carbon is β.

For carbohydrates of L series a) If hydroxyl is up, the configuration of anomeric carbon is α and b) If hydroxyl is down, the configuration of anomeric carbon is β.

Note: A ring containing five carbons and one oxygen is referred pyran

Physical Properties of Glucose:

• It is a white crystalline solid.
• It is soluble in water but sparingly soluble in alcohol.
• As it contains 4 asymmetric carbon atoms, it is an optically active compound.
• It is dextrorotatory. It has a specific rotation of  + 52.5°.

Fructose::

Fructose also has the molecular formula C₆H₁₂O₆ and on the basis of its reactions, it was found to contain a ketonic functional group at carbon number 2 and six carbons in the straight chain as in the case of glucose.

It belongs to D-series and is a laevorotatory compound. It is appropriately written as D-(–)-fructose. Its open chain structure is as shown.

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All those simple or complex organic and inorganic substances found in different living forms are collectively called as biomolecules. Biomolecules can be grouped into two types micro and macromolecules. The micro molecules have moderate to low molecular mass.  e.g. sugars, amino acids, and their derivatives, vitamins, coenzymes, nucleotides, inorganic salts, and water. The macromolecules have very large molecular mass. e.g. carbohydrates, proteins, fats, oils, lipids, and nucleic acids. These biomolecules contribute in one or other way to the structure and/or function of the living system.

Biomolecules are the lifeless molecules which combine in a specific manner to produce life or control biological reactions.  e.g. carbohydrates, proteins, fats, oils, lipids, and nucleic acids.

Carbohydrates are polyhydroxy aldehydes or polyhydroxy ketones or compounds that give polyhydroxy aldehydes or polyhydroxy ketones on hydrolysis. They contain at least one asymmetric carbon atom.   e.g. Glucose (C₆H₁₂O₆), Cane sugar Sucrose (C₁₂H₂₂O₁₁). They are a class of naturally occurring organic compounds found in plants and animal kingdoms. They contain carbon, hydrogen, and oxygen actually carbohydrates mean hydrates of carbon. The proportion of hydrogen and oxygen is definite 2:1. The exception is Rhamnose (C6H12O5). The sources of carbohydrates are Rice, Wheat, Jowar, Bajra, etc. They function as structural material (Cellulose) and reserved food material (Sugar, starch). They are essential for the growth and maintenance of animal life.

Classification of Carbohydrates:

On the Basis of Solubility and Taste:

Based on solubility and the taste, the carbohydrates are classified into two types. a) Sugars and b) Non-sugars.

Characteristics of sugars:

• They are soluble in water.
• They are crystalline in nature.
• They are sweet in taste.
• e.g. Cane sugar, Glucose etc.

Characteristics of non sugars:

• They are insoluble in water.
• They are amorphous in nature.
• They are tasteless
• e.g. Starch, cellulose, etc.

On the Basis of Hydrolysis Behaviour:

Based on hydrolysis behaviour the carbohydrates are classified into three types. a) Mono-Saccharides b) Oligo-Saccharides and c) Poly-Saccharides.

Monosaccharides: 

Carbohydrates which are basic units or which cannot be hydrolyzed further are called monosaccharides.

Characteristics of mono-saccharides:

• Carbohydrates which are basic units or which cannot be hydrolysed further are called monosaccharides.
• They are basic units of carbohydrates.
• They cannot be hydrolysed further into small units.
• They contain six carbon atoms in a molecule.
• They are water-soluble and sweat in taste.
• Depending upon the presence of an aldehydic group or a ketonic group they are further subclassified into aldoses and ketoses respectively.

Aldoses: The monosaccharides containing the aldehydic group are called aldoses. Examples: Aldopentose C₅H₁₀O₅ – Arbaniose, Xylose, Ribose. Aldohexose C₆H₁₂O₆ – Glucose, Galactose

Ketoses: The monosaccharides containing the ketonic group are called ketoses. Examples: Ketopentose C₅H₁₀O₅ – Ribulose.Ketohexose C₆H₁₂O₆ – Fructose.

Oligo-saccharides:

Carbohydrates which on hydrolysis give definite no. of monosaccharides i.e. 2 to 9 molecules of monosaccharides are called oligosaccharides. Depending upon no. of monosaccharides formed on hydrolysis they are further subclassified into Di-saccharides, Tri-saccharides, Tetra-saccharides, etc.

Di-saccharides: 

• Di-saccharides on hydrolysis give two molecules of monosaccharide.
• e.g. Cane sugar (Sucrose) (C₁₂H₂₂O₁₁)on hydrolysis gives one molecule of glucose and one molecule of fructose, Maltose (C₁₂H₂₂O₁₁) on hydrolysis gives two molecules of glucose, Lactose (C₁₂H₂₂O₁₁) on hydrolysis gives one molecule of glucose and one molecule of galactose.
• Disaccharides are crystalline, water-soluble and sweet in taste.
• They have the general formula (C₁₂H₂₂O₁₁).

Tri-saccharides:

• Tri-saccharides on hydrolysis give three molecules of monosaccharide.
• e.g. Raffinose (C₁₈H₃₂O₁₆).

Tetra-saccharides: 

• Tetra-saccharides on hydrolysis give three molecules of monosaccharide.
• e.g. Stachyose (C₂₄H₄₂O₂₁).

Poly-saccharides:

• Carbohydrates which on hydrolysis give indefinite or large no. of monosaccharides (more than 10)  are called polysaccharides.
• They are natural polymeric carbohydrates. They are insoluble in water, amorphous and tasteless.
• They are nonsugars.
• They have the general formula (C₆H₁₀O₅)_n.  e.g. Starch, Cellulose, Inulin, Dextrin etc
• Cellulose is a linear polymer of β-Glucose units while starch is a branched polymer of a-glucose units.

Action of Fehling’s solution on Sucrose:

• Sucrose gives polyhydroxy aldehydes on hydrolysis.
• Fehling’s solution is a mild oxidising agent. It is a complex of cuprous ions of tartaric acid in the presence of an alkali.
• When treated with hydrolysis products of sucrose, the aldehyde formed gets oxidised and the red precipitate of cuprous oxide is formed.

Fischer Projection:

The wedge and dash representations of stereochemistry can often become cumbersome, especially for large molecules which contain a number of stereocenters. An alternative way to represent stereochemistry is the Fischer Projection, which was first used by the German chemist Emil Fischer. The Fischer projection represents every stereocenter as a cross. The horizontal line represents bonds extending out of the plane of the page, whereas the vertical line represents bonds extending into the plane of the page.

When working with Fischer Projections, keep in mind the following rules:

• Because the “up” and “down” aspects of the bonds don’t change, a Fischer projection may be rotated by 180 degrees without changing its meaning.

• A Fischer projection may not be rotated by 90 degrees. Such a rotation typically changes the configuration to the enantiomer.

• To find the enantiomer of a molecule drawn as a Fischer projection, simply exchange the right and left horizontal bonds.

• To determine whether the molecule in Fischer projection is a meso compound, draw a horizontal line through the center of the molecule and determine whether the molecule is symmetric about that line.

• D – and L – Sugars: Glyceraldehyde is an aldotriose it is the simplest optically active compound. It contains one asymmetric carbon atom and has two enantiomers (stereoisomers) as shown

• In Fischer projection, if the hydroxyl group at the lowest chirality centre points to the right, the monosaccharide is referred as D- sugar. if the hydroxyl group at the lowest chirality centre points to the left, the monosaccharide is referred as D- sugar.

Structure of Some Monosaccharides:

Triose (C₃H₆O₃):

Tetrose (C₄H₈O₄):

Pentose (C₅H₁₀O₅):

Hexose (C₆H₁₂O₆):

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In this article, we shall study structural units of nucleic acid called nucleotides.

In 1869, Friedrich Miescher separated cellular substance from the nuclei of pus cell and called it ‘Nuclein’. Due to acidic nature, the substance is further called as nucleic acid. There are two types of nucleic acids a) Deoxyribonucleic acid (DNA) found primarily in the nucleus of cells and b) Ribonucleic acid (RNA) found mainly in the cytoplasm of living cells.

Chemical Components of Nucleic Acids:

Nucleotides:

Nucleotides are the structural units of nucleic acids. Each nucleotide has three components

Sugars:

The five-carbon sugar (pentose) in nucleic acids is ribose or a ribose derivative. It has a pentagonal ring structure. In RNA the sugar is ribose, in DNA it is 2-deoxyribose.

The only difference between these two sugars is found at the 2-carbon of the ribose ring. Ribose has a hydroxyl group (-OH) bound to this carbon, while deoxyribose has a hydrogen atom (“deoxy” means no oxygen).

Phosphate Group:

The second component of a nucleotide is derived from phosphoric acid (H₃PO₄).

Phosphoric acid contains three hydroxyl groups attached to phosphorous.

Phosphoric acid             Phosphate group

From these three OH groups, two are responsible for strand formation.

Nitrogen or Organic Bases:

The organic bases found in nucleic acids are derivatives of pyrimidine or purine.

Pyrimidine is a six-membered heterocyclic ring. A heterocyclic ring is a ring compound containing atoms that are not all identical. Purine is a fused ring compound containing a six-membered ring connected to a five-membered ring.

Pyrimidines:

There is only one ring which is hexagonal and heterocyclic. The ring consists of four carbons and three nitrogens with an alternate single and double bond. Numbering is done clockwise starting from nitrogen. Nitrogen atoms are present at the first and third positions. Rest positions are occupied by carbon. Such a ring is called a pyrimidine ring.

The three pyrimidine derivatives found in nucleic acids are cytosine (C), thymine (T), and uracil (U).

Cytosine = 2-oxy-4-amino pyrimidine

Thymine = 2,4-dioxy-5-methyl pyrimidine

Uracil = 2,4-dioxy pyrimidine

Characteristics of Pyrimidines:

• They are single ring compounds.
• They are formed by a pyrimidine ring.
• There are 4 carbons and 2 nitrogens in the ring.
• Nitrogen atoms are present at the first and the third position.
• Oxygen is attached to second carbon by a double bond.
• A glycosidic bond is formed between nitrogen at the first position in pyrimidine and carbon at the first position in pentose sugar.

Purines:

There are two rings (dicyclic) in this nitrogen compound. There are nine atoms in the molecule of which 4 are nitrogen and 5 are carbon atoms. There are 6 atoms in the first ring called pyrimidine ring and 5 atoms in the second ring called imidazole ring. Atoms are numbered anticlockwise in pyrimidine ring and clockwise in the imidazole ring.  The imidazole ring.is fused with pyrimidine ring at the 4th and 5th position so that the two rings share carbon atom at 4th and 5th position. The nitrogen is present at first, third, seventh and ninth position in the ring.

The two purine derivatives found in nucleic acids are adenine (A) and guanine (G).

Characteristics of Purines:

• They are double ring compounds.
• They are formed by pyrimidine and imidazole ring.
• There are 5 carbons and 4 nitrogens in the ring.
• Nitrogen atoms are present at the first, third, seventh and ninth position.
• No oxygen is attached to the second carbon.
• A glycosidic bond is formed between nitrogen at the ninth position in pyrimidine and carbon at the first position in pentose sugar.

Note:

• Adenine, guanine, and cytosine are found in both DNA and RNA. Thymine is found only in DNA, while uracil is found only in RNA.
• Thymine and uracil are often used to differentiate DNA from RNA.

Nucleosides:

When ribose or 2-deoxyribose is combined with a purine or pyrimidine base, then the combination is called nucleoside. A nucleoside is basically a nucleotide that is missing the phosphate portion.

Thus Nucleoside = Sugar + Nitrogen Base

In a nucleoside, the pentose sugar and base are joined by an N-glycosidic bond formed between semialdehyde -OH group of monosaccharide at 1 and H of the pyrimidine base at N-1 or the purine base at the 9th nitrogen atom of the ring

New Naming System for Nucleosides:

Nucleotides:

The nucleotides are named according to their nitrogenous base. For e.g. a nucleotide containing thymine is called thymine nucleotide.

Thus Nucleotide = Pentose Sugar + Nitrogen Base + Phosphate Group

or Nucleotide = Nucleoside + Phosphate Group

New Naming System for Nucleotides:

Linking of Nucleotides in Polynucleotides:

A polynucleotide chain is formed by connecting several nucleotides in succession. Several thousand nucleotides are linked together by 3′-5′ phosphodiester bond in which the phosphate group carried in 5th carbon atom of pentose in one nucleotide is linked to 3′ hydroxyl group of 3′ carbon of the pentose of the next nucleotide. These bonds provide considerable stiffness to polynucleotide chain.

The bond is called phosphodiester bond because one molecule of phosphoric acid joins with sugar molecules of two nucleotides through an ester linkage.

Joining two nucleotides is called dinucleotide, joining three nucleotides is called trinucleotide and so on. A chain up to joining of twenty nucleotides is called oligonucleotide. If there is joining of more than twenty nucleotides it is called polynucleotide.

RNA is a polynucleotide that, upon hydrolysis, yields D-ribose, phosphoric acid, and the four bases adenine, guanine, cytosine, and uracil.

DNA is a polynucleotide that yields D-2′-deoxyribose, phosphoric acid, and the four bases adenine, guanine, cytosine, and thymine.

The Directionality of Polynucleotide Chain:

Adjacent nucleotides in a single strand of the polynucleotide are joined by a phosphodiester bond between their 3′ and 5′ carbons. This means that the respective 5′ and 3′ carbons are exposed at either end of the polynucleotide, which are therefore called the  5′-P end and the 3′-OH end. These are also called the phosphoryl (5′-P terminus) and hydroxyl (3′-OH terminus) ends, respectively, because of the chemical groups typically found at those ends.

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DNA as Genetic Material

Griffith Experiment:

Background:

Meischer isolated nuclein from nuclei of WBCs in 1869. Walter Sutton, Thomas Hunt Morgan established that the hereditary material lies in the nucleus in chromosomes. Chromosomes are formed of proteins and nucleic acid, DNA. For many years proteins were assumed to be the carrier of hereditary information due to their structural and functional diversity. By 1926 the mechanism for genetic inheritance had reached the molecular level. But exactly which molecule is responsible for heredity was not confirmed.

Bacterium Streptococcus pneumoniae occurs in two strains:

• Smooth Virulent Strain (S-III): The smooth virulent strain of Streptococcus pneumoniae is enclosed in polysaccharide capsule. Due to the presence of the capsule their colonies are smooth and shiny. Hence they are called smooth strain (S). This capsule protects them by preventing them engulfed by WBCs. As they are not destroyed by WBCs, they cause pneumonia in mice.
• Rough Avirulent Strain (R-II): The rough avirulent strain of Streptococcus pneumoniae lacks polysaccharide capsule and hence are destroyed by WBCs. Due to the absence of the capsule, their colonies have an irregular appearance. Hence they are called rough strain (R). As they are destroyed by WBCs they do not produce symptoms of pneumonia in mice.

Experiment:

In 1928 Frederick Griffith, in a series of experiments with Diplococcus pneumoniae (bacterium responsible for pneumonia), witnessed a miraculous transformation in the bacteria. During the course of his experiment, the bacteria (living organism) had changed in physical form.

The pneumococcus bacterium occurs naturally in two forms with distinctively different characteristics. The virulent or pathogenic (S-strain) form has a smooth polysaccharide capsule that is essential for infection. The nonvirulent or nonpathogenic (R-strain) lacks the polysaccharide capsule, giving it a rough appearance.

Step – 1: S-type of the pneumococcus bacteria were injected into healthy mice. The mice were infected and died from pneumonic infection within a few days,

Step – 2: R-type of the pneumococcus bacteria were injected into healthy mice. The mice were not infected and continue to live.

Step – 3: Heat Killed S-type of the pneumococcus bacteria were injected into healthy mice. The mice were not infected and continue to live.

Step – 4: A mixture of heat-killed S-type and live R-type pneumococcus bacteria were injected into healthy mice. It produced lethal results. The mice died. On observation, Griffith discovered a mixture of R-Type and living forms of the S-type bacteria in the infected dead mice.

Conclusions:

Griffith hypothesized that something has transformed the non-lethal R-type avirulent bacteria into lethal S – Type virulent bacteria. The heat-killed S-strain bacteria should be responsible for it. This transformation is called Griffith effect or bacterial transformation.

Some “transforming principle”, enabled the R-strain to synthesize a smooth polysaccharide coat and become virulent. He further observed that the “transforming principle” was transferred to the next generation. Thus “transforming principle” should be genetic material. Further, it was proved that the “transforming principle” referred to by Griffith is DNA.

Avery, Macleod and McCarty Experiment:

In 1944  Oswald Avery, Collin Macleod and Maclyn McCarty performed the same experiment as that by Griffith but their aim was definite to locate the factor responsible for a transformation of non-lethal R-type bacteria into lethal S – Type bacteria. They used a test tube assay instead of mice.

They purified DNA, RNA, proteins and other materials from heat-killed S – type bacteria using corresponding dissolving enzymes. Then they mixed purified content with R – type to see which one could transform living R – type into S – type.

Only those mixed with DNA were transformed into S – type bacteria. When DNA was treated with Deoxyribonuclease, the DNA was digested and dissolved, there was no transformation of R-type bacteria into S – Type bacteria. This confirmed that  “transforming principle” is DNA. But scientist community at that time was not convinced.

Hershey – Chase Experiment:

Alfred Hershey and Martha Chase (1952) experimentally proved that DNA is the only genetic material. They worked with viruses that infect bacteria called bacteriophages (T2-phages).

The bacteriophage attaches by its tail to the bacteria and its genetic material then enters the bacterial cell and protein coat is left outside. The bacterial cell treats the viral genetic material as if it was its own and subsequently produces more virus particles. A large number of phage-DNA molecules are formed. Each of these DNA molecules develops its own protein coat forming daughter phage particles.

Hershey and Chase performed an experiment to discover whether it was protein or DNA from the viruses that entered the bacteria.

Step – 1: 

They used the fact that DNA contains phosphorus but not sulphur, while protein contains sulphur but not phosphorous. They grew some viruses on a medium that contained radioactive phosphorus (³²P) and some others on the medium that contained radioactive sulphur (³⁵S).

Observations: Viruses grown in the presence of radioactive phosphorus contained radioactive DNA but not radioactive protein. Similarly, viruses grown on radioactive sulphur contained radioactive protein but not radioactive DNA.

Step – 2:

Radioactive phages were allowed to attach to E. coli bacteria. As the infection proceeded, the viral coats were removed from the bacteria by agitating them in a blender and the virus particles were separated from the bacteria by spinning them in a centrifuge.

Observations:

Bacteria which was infected with viruses that had radioactive DNA were radioactive, indicating that DNA was the material that passed from the virus to the bacteria. The phages grown in radioactive phosphorous passed their radioactivity to the daughter phage particles through DNA.

Bacteria that were infected with viruses that had radioactive proteins were not radioactive. The phages grown in radioactive sulphur did not pass their radioactivity to the daughter phage particles through proteins. This indicates that proteins did not enter the bacteria from the viruses.

Conclusion:

Therefore DNA is the genetic material that is passed from virus to bacteria.

RNA as Genetic Material

Frankel-Conrat and Singer Experiment:

H. Frankel-Conrat and B. Singer (1957) performed an experiment with tobacco mosaic virus (TMV) and demonstrated that in some cases RNA acts as a genetic material.

Tobacco mosaic virus (TMV) does not contain any DNA. It consists of RNA surrounded by a hollow cylinder of protein subunits. They found that the virus could be broken into component parts and they could again be reassembled or reconstituted to form a functional virus.

Viruses with the single-stranded genome (RNA) use a single strand as a template and synthesize a complementary single strand of DNA. This complementary single-strand DNA, in turn, synthesize its complementary strand and forms a double-stranded DNA.

Techniques were first developed for separating TMV particles into RNA and proteins. Later by using RNA and proteins separately in tests for infectivity, it could be shown that RNA alone was able to cause infection. Such property was not found in the protein fraction.

When the cell debris (protein coat) of the virus was introduced into tobacco leaf, the leaf remained healthy. When the cell filtrate (nucleic acid) was injected into tobacco leaf, it was infected with the virus and died. This shows that the RNA is causing the infection and not the protein.

The progeny viruses produced were always found to be phenotypically and genotypically identical to the parent strain from which the RNA had been obtained.

In one experiment, two viruses used were tobacco mosaic virus (TMV) and Holmes rib-grass virus (HRV). Reciprocal hybrid using RNA of one strain and protein of the other strain is obtained. It was found that when these hybrids were used for infection, the progeny had proteins which corresponded to the virus from which RNA of the infecting virus particles was derived.

Properties of DNA in Genetic Material:

• DNA has the ability to store hereditary information in coded form.
• DNA is present in all the cells of the organism.
• DNA shows diversity corresponding to the varieties existing in the organisms.
• DNA has the capacity to replicate itself to produce a carbon copy that could be transferred to daughter cells (successive generations).
• DNA is able to express itself through specific biological molecules like proteins and enzymes.
• DNA has physical and chemical stability so that the stored information is not lost.
• DNA (genes) is capable of differential expression so that the various parts of an organism may acquire specific form, structure and functions in-spite of having the same genetic material.
• DNA (genes) undergoes gradual mutations and recombinations so that the new characters appear in the organism to produce diversity.

Comparision Between DNA and RNA as Genetic Material:

• DNA is the genetic material in most organisms except in plant viruses and some animal viruses where RNA acts as genetic material.
• Both have a stable structure and yet capable of undergoing mutations (slow changes).
• Both are capable of transcription and translation.
• As both DNA and RNA follow base pair-rule and hence exhibit complementarity. Both of them have the ability to direct their duplication.
• DNA is very stable while RNA is more reactive (less stable).
• RNA mutates faster than DNA
• RNA can code for the synthesis of protein directly while DNA depends on RNA to transfer the message of protein synthesis from the nucleus into the cytoplasm.
• From the above points, we can conclude that DNA is more stable. Hence are more suited for storing genetic information.

Science > Biology > Gene its Nature, Expression and Regulation > Genetic Material

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In the last article, we have studied the meaning of the term gene. In this article, we shall study types of genes.

Based on the function and activity, the genes are classified as follows.

Housekeeping Genes or Constitutive Genes:

Housekeeping genes are involved in basic cell maintenance and, therefore, are expected to maintain constant expression levels in all cells and conditions. They are functional in all types of body cells of a multicellular organism and all the time. They are required for basic cellular activity. They are not regulated.

Example: Genes associated with glycolysis are active in all types of cells and all the time throughout life.

housekeeping genes are instrumental for calibration in many biotechnological applications and genomic studies. Advances in our ability to measure RNA expression have resulted in a gradual increase in the number of identified housekeeping genes.

Luxury Genes or Noncontitutive Genes:

These genes are not always expressing themselves in a cell. They remain inactive for most of the time in the lifespan of an individual and is expressed in certain cells or at a certain time only when their products are needed. These are called luxury genes or specialist genes.

Humans comprise approximately 200 different types of cells, such as skin cells, liver cells, and nerve cells. Each cell varies in both the structure and the function because different sets of genes are expressed in each of them. For example, the serum albumin gene is expressed only in hepatocytes (liver cells), while the insulin gene is expressed only in pancreatic beta cells. They are switched on or off according to the requirement of cellular activities.

Example: the gene for nitrate reductase in plants, lactose system in Escherichia coli. There are some genes in the human body which are present in all the body cells but some are functional in kidney cells, some in liver cells and some in intestine or stomach. They are associated with adaptive enzyme synthesis.

Luxury genes are of further classified as inducible and repressible. The genes are switched on in response to the presence of a chemical substance or inducer which is required for the functioning of the product of gene activity are called inducible genes, e.g., nitrate for nitrate reductase. The genes which continue to express themselves till a chemical (often an end product) inhibits or represses their activity are called repressible genes.

Structural Genes (Cistrons):

These genes code for chemical substances which contribute to the morphological or functional trait of the cell. These are called cistrons. They are continuous in prokaryotes and split into introns and exons in eukaryotes. They are further classified as

• Polypeptide-coding Genes: These genes code for mRNAs which in turn code for polypeptides. The polypeptide produced may act as a component of an organelle (as actin of muscle fibre); an enzyme (as DNA polymerase); a transport protein (as haemoglobin); a hormone (as insulin); a receptor or carrier protein of cell membrane; an antibody, an antigen.
• Polyprotein-coding Genes: These genes code for more than one polypeptide per gene.
• RNA-coding Genes: These genes code for rRNAs and tRNAs.

Regulator Genes:

These genes code for repressor proteins for regulating the transcription of cistrons.

Operator Genes:

An operator gene acts as a switch to turn on or off the transcription of a structural gene as and when required by the cell.

Promoter Genes:

These genes are DNA sequences (sites) for the binding of RNA polymerase for the transcription of RNAs by the structural genes.

Terminator Genes:

These genes are DNA regions (lying t end of message) where RNA polymerase activity stops to suspend transcription of structural genes.

Uninterrupted Genes or Continuous Genes Or Collinear Genes:

In prokaryotes, the sequence of nucleotides in the gene corresponds exactly with the sequence of amino acids in the protein. Such nucleotide sequence codes for a particular single polypeptide chain.  Each gene is a continuous stretch of DNA whose length is related to the size of protein to be synthesized. Thus these genes and proteins are collinear.

Interrupted Genes or Discontinuous Genes or Split Genes:

Generally, a gene has a continuous sequence of nucleotides. However, it was observed that the sequence of nucleotides was not continuous in the case of some genes, the sequences of nucleotides were interrupted by intervening sequences. Such genes with the interrupted sequence of nucleotides are called split genes or interrupted genes. Thus, split genes have two types of sequences, viz., normal sequences and interrupted sequences

The Concept of Exons and Introns:

The coding units containing biological information are called exons. and intervening non-coding DNA segments are called introns. Introns are present in the genes of eukaryotes, viruses, and archaebacteria. Interrupted genes produce the primary transcript RNA. It acts as a precursor as it is a faithful copy of the interrupted gene.

The functional RNA is formed by the removal of introns and rejoining exons. This process is known as RNA splicing.

Overlapping Genes or Alternate Genes:

A few genes in certain bacteria and animal viruses code for two different polypeptides (more than one protein). These are called overlapping genes. In this case, the specific sequence is shared between two non-homologous proteins. In these genes, the first and second half of the gene codes fora specific protein that represents the first or second half of the protein, specified by the full gene.

Alternative Genes:

The concept of alternative genes was given by Gilbert and is known as Gilbert hypothesis. They are formed when exons from different discontinuous genes get connected forming several new combinations. These genes produce proteins in which one part is common while another part is different.

Jumping Genes or Transposons: 

They are segments of DNA that can jump or move from one place in the genome to another. Transposons were first discovered by Nobel prize winner Mc Clintock (1951) in the case of Maize when she found that a segment of DNA can move from one position to another in the genome of the cell. Recently they have been described in snapdragon, Drosophila, mice, and bacteria.

Transposons possess repetitive DNA, either similar or inverted, at their ends. The two major events took place during transposition. There is a duplication of the target sequence in the recipient DNA molecule and the insertion of transposons between the repeated target sequences.

Gene Families and Pseudogenes:

They are genes which have homology to functional genes but are unable to produce functional products due to intervening nonsense codons, insertions, deletions, and inactivation of promoter regions, Pseudogenes are genomic DNA sequences similar to normal genes but non-functional; they are regarded as defunct relatives of functional genes.

Most of the prokaryotic genes are represented only once in the genome. But many eukaryotic genes are presented in multiple copies. These multiple copies of genes are called gene families or pseudogenes. They may be clustered in the same region of DNA or dispersed to different chromosomes.

e.g., several of snRNA genes.

Single Copy Genes:

The genes are present in single copies (occasionally 2-3 times). They form 60-70% of the functional genes. Duplications, mutations and exon reshuffling between two genes form new genes.

Processed Genes:

They are eukaryotic genes which lack introns. Processed genes are generally nonfunctional as they lack promoters.

Multi-genes (Multiple Gene Family):

It is a group of similar or nearly similar genes for meeting the requirement of time and tissue-specific products.

Science > Biology > Gene its Nature, Expression and Regulation > Types of Genes

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In this article, we shall the essential characters of genetic material, the meaning of the term gene, its characteristics, and its functions.

Essential Features of Genetic Material:

• It should have the ability to store hereditary information in coded form.
• It should be present in all the cells of the organism.
• It should show diversity corresponding to the varieties existing in the organisms.
• It should have the capacity to replicate itself to produce a carbon copy that could be transferred to daughter cells (successive generations).
• It should able to express itself through specific biological molecules like proteins and enzymes.
• It should have physical and chemical stability so that the stored information is not lost.
• It should be capable of differential expression so that the various parts of an organism may acquire specific form, structure and functions in-spite of having the same genetic material.
• It should undergo gradual mutations and recombinations so that the new characters appear in the organism to produce diversity. Thus The genetic material should be able to generate its own kind and also new kinds of molecules.

Gene:

A gene may be defined as a segment of DNA which is responsible for inheritance and expression of a particular character. A gene is a segment of DNA that provides instructions for the synthesis of a specific protein or a particular type of RNA.

Mendel was first to call genes as a unit of inheritance and called them factors. The term ‘gene’ was derived from the Greek word ‘Genesis’ which gives the meaning ‘to be born’ and was coined by a Danish Geneticist- Wilhelm Johannsen in 1909.

Characteristics of Genes:

• Genes are the functional unit of heredity, variation, mutation and evolution. Genes determine the physical as well as physiological characteristics of organisms. Genes are responsible for transferring these characters from parents to the offspring generation after generation.
• They are situated in chromosomes.
• Every gene occupies a fixed position in a chromosome. This position is called a locus.
• They are arranged in a single linear order in a chromosome as beads on a string.
• They express them by the synthesis of proteins and enzymes, which control cell metabolism. Thus they determine the physical and metabolic characteristics of the cell. Each gene synthesizes a particular protein which acts as an enzyme and brings about the appropriate change.
• They can produce a duplicate copy of themselves. The process is called replication.
• In a single gene they may occur in several different forms called alleles. Only those genes are known which have their alternative alleles. The alleles may be related as dominant or recessive but not always.
• Some alleles mutate more than once and have more than two alleles. These alleles are known as multiple alleles. Whatever may be the number of alleles in a multiple series only two of them are found in an individual because of the presence of two homologous chromosomes of each type.
• They may show a sudden change in expression from one form to another due to a change in composition. This sudden change is called mutation and the new allele is called a mutant.
• There is a large number of genes in organisms while the number of chromosomes is small. Hence several genes are located in each chromosome. In the human being, there are about 40,000 known genes located on 23 chromosomes.
• A gene is a segment of DNA which contain information for the synthesis of one enzyme or one polypeptide chain coded in the language of nitrogenous bases or the nucleotides.

Modern Concept of Gene:

Seymour Benzer in 1955 introduced the terms cistron, muton, and recon

Cistron (Unit of function):

• It is a segment of DNA having information of synthesis of particular protein or RNA.
• It is responsible for the expression of a trait.
• It can be several bp (base pairs) long.

Muton (Unit of mutation):

• It is a segment of DNA that can undergo mutation.
• It consists of few nucleotides (one to a few bp long).

Recon (Unit of recombination):

• It is a segment of DNA that participates in recombination through crossing over during meiosis.
• It consists of a few to many base pairs.

Operon: 

• It is a combination of an operator gene, a structural gene or sequence of structural genes which act together as a unit.

Replicon:

• It is the unit of replication

Functions of Genes:

• Genes are the functional unit of heredity, variation, mutation, and evolution. Genes determine the physical as well as physiological characteristics of organisms. Genes are responsible for transferring these characters from parents to the offspring generation after generation.
• Genes control the phenotypes of the offspring including both the structural and functional characters.
• Genes control reproduction through their replication.
• Genes undergo mutations and produce polymorphism and variations in the individuals of a population. These mutations are also associated with metabolic disorders and inborn errors of metabolism.
• Genes are associated with the aging process.
• Genes are responsible for producing cancer.
• Control genes regulate transcription of mRNA and thus regulate the amount of protein synthesized.
• They code for different types of RNAs other than mRNA like rRNA and tRNA.
• Genes are responsible for switching on and off specific genes as per the requirement of the organism.
• Genes control the functioning of luxary genes.
• They produce cellular differentiation during development.

Science > Biology > Gene its Nature, Expression and Regulation > Gene: The Concept, Characteristics, and Functions

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