DNA replication is a complex process requiring a number of enzymes and other proteins, and even the participation of RNA. The entire process of replication involves the following steps, and these are illustrated in Figure 2.19. Parent Replica Guanine Cytosine Adenine Thymine 3¢ 3¢ 5¢ 5¢ 5¢ 3¢ 3¢ 5¢
It is important to see that the sequence of nucleotides has a direction or polarity, with one free 5¢-end and the other terminus possessing a 3¢-end not coupled to another nucleotide. The two strands of DNA have opposite polarity: they run 5¢®3¢ in opposite directions. Combining this information with the base pairing rules, we can infer from the nucleotide sequence of a single deoxyribonucleotides chain, for example 5¢ CGAATCGTA 3¢, that the corresponding fragment of an intact, double-stranded DNA molecule looks like the following:
5¢ CGAATCGTA 3¢ 3¢ GCTTAGCAT 5¢
Thus, in an informational sense, knowledge of one strands sequence implies the sequence of the complimentary strand. Each strand is a template for the other. This feature of DNA provides directions for the synthesis of daughter DNA from a parent DNA molecule, and the process is called DNA replication. If the two complimentary strands are separated and double helices are constructed from each strand following the base pairing rules, the end products are two new molecules, each identical to the original double- stranded DNA, and each containing one new strand and one old strand.
Thus, the biological message is coded in the DNA nucleotide sequence. As the parent strands separate, complimentary strands are added to each parent, resulting in two daughter molecules identical to the parent.
Finally, each daughter molecule contains one strand from the parent. Regeneration of DNA from original DNA segments is known as DNA replication and it is semi-conservative as shown in Figure 2.20.
Figure 2.20 Semi-conservative replication of DNA. Parental DNA
2nd Generation Ist Generation
Based on the findings of Watson and Crick, the central dogma of the molecular genetics is understood and the flow of information is seen to be essentially in one direction from DNA to the protein.
Three major steps are defined in the flow of information. They are replication, transcription and translation of the genetic material. The same is shown in Figure 2.21.
We have already discussed the replication of DNA. Once the replication occurs, the DNA has the information stored in it. Later on it undergoes a series of changes as follows.
Transcription: Transcription is the copying of a complementary messenger
RNA strand on a DNA template (one of the strands of DNA on which mRNA is transcribed). The process of transcription requires the template, high- energy compounds like ATP, GTP, the enzyme RNA polymerase and divalent cations. The enzyme RNA polymerase consists of a core enzyme with subunits and sigma factor (s). The sigma factor initiates the transcription of mRNA on the DNA template and the core enzyme continues transcription.
Translation: In this the genetic information present in mRNA directs the
order of specific amino acids to form a polypeptide or a protein. The translation process consists of activation of amino acids, transfer of activated amino acids to tRNA, initiation of polypeptide chain, chain elongation and termination. These processes require ribosomes, mRNA and tRNA, initiation factors, termination factors, and high energy compounds like ATP, GTP, and the divalent ions like Mg2+.
Separation of double-stranded DNA by heat is an important method in DNA characterization. Because AT base pairs involve two hydrogen bonds and GC base pairs have three, AT-rich region of DNA melt (i.e. the two strands separate) before GC-rich regions. The melting process is readily monitored by following absorbance of the DNA solution at 260 nm: single- stranded DNA absorbs more strongly, so that DNA melting is measured as an increase in overall absorbance. The melting temperature Tm is the
temperature at which the absorbance is midway between the fully double- stranded and completely melted limits.
Figure 2.21 Information flow and storage in cell systems with major processes involved. Transcription
DNA having stored information
RNA transfer information by mRNA Translation
Protein synthesis in ribosomes (Function)
If a solution of melted DNA is cooled, the separated complimentary strands will anneal to reform the double helix. Similarly, if two different single-stranded segments of DNA have complimentary base pair sequences, these will hybridize to form a double-stranded segment. Also, double-stranded DNA in solution behaves hydrodynamically like a rigid rod while single- stranded DNA acts like a randomly coiled polymer.
2.8 CARBOHYDRATES
Carbohydrates are the most abundant organic substances in nature. Sugars, starches and cellulose found in green plants, glycogen in animal tissues, and glucose in the body fluids of animals are all examples of carbohydrates.
They occur in food, wood, paper and synthetic fibres. Carbohydrates have an empirical formula Cn(H2O)n where n ³ 3. They are also called saccharides meaning sugars.
Carbohydrates play a key role as storage and structural compounds in the cell. They also play crucial roles in modulating aspects of chemical signalling in animals and plants. Carbohydrates are synthesized by plants through photosynthesis as illustrated in Figure 2.22.
Figure 2.22 Synthesis of carbohydrates.
CO2 and H2O are converted through photosynthesis into sugars in the
presence of sunlight and are then polymerized to yield polysaccharides. Carbohydrates are classified into (a) monosaccharides, (b) disaccharides, and (c) polysaccharides.
2.8.1 Monosaccharides
Monosaccharides are the simplest and smallest carbohydrates and contain three to nine carbon atoms, and are the building blocks of complex carbohydrates. These cannot be hydrolyzed into a simpler sugar, for example, glucose which has a molecular formula of C6H12O6.
CH2O + O2
Sunlight (Energy absorbed)
CO2+ H2O Photosynthesis
Respiration
Glucose may be present in the form of a linear or ring structure. In solution, D-glucose is in the form of a ring structure or pyranose. The L-form has a minor role in the biological systems.
Two conventional ways of representing the structure of glucose are as shown in Figure 2.23. CHO HCOH OHCH HCOH HCOH CH OH2 D-Glucose, linear structure C HCOH OHCH HCOH HC CH OH2 D-Glucose ring structure (pyranose) O OH H
The structures of a-D-glucose and b-D-glucose are as shown in Figure 2.24.
Figure 2.24 Structures of a-D-glucose and b-D-glucose.
O OH OH OH OH CH OH2 O CH OH2 OH OH OH OH b-D-Glucose a-D-Glucose
According to the number of carbon atoms, the monosaccharides are classified into trioses (3C), tetroses (4C), pentoses (5C), and hexoses (6C).
Trioses (C3H6O3): Trioses include glyceraldehydes and dihydroxyacetone.
They are the intermediates in respiration during the cycle of glycolysis, photosynthesis (dark reaction) and other branches of carbohydrate metabolism.
glyceraldehydes ® glycerol ® triglyceride (lipid)
D-Glucose ring structure (pyranose) D-Glucose, linear
structure
Tetroses (C4H8O4): Tetroses like erythrose are rare in nature and occur
mainly in bacteria.
Pentoses (C5H10O5): Pentoses include ribose, ribulose, etc. In the synthesis
of nucleic acids, ribose is a constituent of RNA while deoxyribose in the case of DNA (Figure 2.25). Pentoses are involved in the synthesis of some coenzymes, e.g. NAD, NADP, coenzyme A, FAD, etc. and also in the synthesis of polysaccharides called pentosans.
Figure 2.25 Structures of ribose and deoxyribose.
OH OH OH 1 2 3 4 OH OH CH OH2 CH OH2 O O 1 2 3 4 5 5 D-Ribose Deoxyribose
Hexoses (C6H12O6): Glucose, fructose, galactose, mannose, etc. are some
examples of hexoses. These are the sources of energy when oxidized in the process of respiration. Of these, glucose is the most common respiratory substrate monosaccharide, involved in the synthesis of disaccharides. If 210 monosaccharides are combined then these are termed as oligosaccharides.
Aldoses and ketoses are the other forms of monosaccharides. In monosaccharides, all the carbon atoms except one have a hydroxyl group attached. The remaining carbon atom is either part of an aldehyde group in which case the monosaccharide is called an aldose or aldo sugar, e.g. ribose, glucose, mannose, galactose, etc. or is part of a keto group, when it is called a ketose or keto sugar, e.g. ribulose, fructose, etc.
2.8.2 Disaccharides
These are formed by the condensation of two monosaccharides usually hexoses. On hydrolysis, a disaccharide yields the two respective monosaccharides.
glucose + fructose ® sucrose (cane sugar) glucose + galactose ® lactose (milk sugar) glucose + glucose ® maltose (in germinating seeds)
Maltose is formed by the condensation of two glucose molecules via a-1,4 glycosidic linkage, which is shown in Figure 2.26.
CH2OH CH2OH
In the same way, sucrose and lactose synthesis are also shown in Figure 2.27. Sucrose is a disaccharide of a-D-glucose, and b-D-fructose, and lactose is a disaccharide of b-D-glucose and b-D-galactose.
Figure 2.26 Formation of a-maltose. O OH OH OH OH CH OH2 O CH OH2 OH OH OH OH O OH OH OH CH OH2 O CH OH2 O OH OH OH 1 4
Figure 2.27 Synthesis of sucrose and lactose.
OH OH CH OH2 O O OH OH OH OH CH OH2 O OH OH OH CH OH2 CH OH2 O OH OH OH O CH OH2 O a-D-Glucose (a) Sucrose b-D-Fructose (b) Lactose b-D-Glucose b-D-Galactose
In general, monosaccharides and disaccharides are crystalline, water soluble, sweet to taste, and are therefore termed sugars.
2.8.3 Polysaccharides
Polysaccharides are normally amorphous, insoluble in water and are tasteless. These are referred to as non-sugars. Polysaccharides are composed of ten to many thousands of monosaccharides in their macromolecules, and their empirical formula is (C6H10O5)n.
Polysaccharides are formed by the condensation of more than two monosaccharides by glycosidic linkages or bonds. Thus, these are the
a-Maltose a-D-Glucose +H2O CH2OH CH2OH CH2OH H2O CH2OH
polymers of monosaccharides. Polymers of pentoses are called pentosans, polymers of hexoses are called hexosans and the polymers of glucose are glucosans. Chemically the polysaccharides are of two typeshomopoly- saccharides and heteropolysaccharides. Homopolysaccharides (homoglycans) are the ones that yield on hydrolysis, a single monosaccharide. Starch, inulin, pectin and chitin are homopolysaccharides. Heteropolysaccharides are the ones that produce a mixture of monosaccharides. Examples include hyaluronic acid, chondroitin sulphates, heparin, hemicellulose, some gums and mucilages.
Homopolysaccharides: Let us learn more about the following homopolysac-
charides.
Starch: It occurs in grains, roots of plants, etc. Starch can be
hydrolyzed into many monosaccharides molecules. The compact structure of a polysaccharide makes it ideal as a storage carbohydrate. Starch has two components, amylose and amylopectin. Amylose has a straight chain structure consisting of several thousands of glucose residues, though the chain coils helically into a more compact shape. Amylopectin is also compact as it has many branches formed by 1,6-glycosidic bonds. Starch deposits are usually about 1030% amylose and 7090% amylopectin. Figure 2.28 shows the glycosidic linkage in amylopectin.
Figure 2.28 Glycosidic linkage in amylopectin.
Glycogen: It is the main polysaccharide occurring in the animal
tissues, particularly in liver and muscle. It consists of a long profusely branched-chain of glucose molecules and is considered to be animal starch. Glycogens form branches similar to the branches seen in starch. The molecular weight of a typical glycogen molecule is less than 5×106 daltons. Cellulose: This is a structural polysaccharide containing a large
number of glucose units arranged in a linear fashion. It is unbranched chain of D-glucose with a molecular weight between 50,000 and 1 million daltons. The linkage between glucose monomers in cellulose is a b1,4 glycosidic linkage (Figure 2.29). O C O C O O O C O CH2 O O O O C O O O O O O
Dextrans: These are produced by yeasts and bacteria and are made up
of glucose residues linked mainly by 1 ® 6 a glucosidic bonds. Branches are formed by occasional 1 ® 4 a linkages and rarely through 1 ® 3 aÿlinkages. They have the property of absorbing water to form viscose colloidal solution. They are not metabolized by the tissues and hence are used for retaining water in circulation for a long period by administering it intravenously (plasma volume extender).
Inulin: Inulin is a low molecular weight polysaccharide present in
tubers. It is a polymer of fructose. Not being utilizable, it is used in assessing the glomerular filtration rate (GFR) in the study of human kidney function.
Agar: Agar is found in sea weeds. It consists of two main components: agarose and agaropectin. Agarose is a non-sulphated linear polymer consisting of alternating residues of D-galactose and 3,6-anhydro-L-galactose. Agaropectin is a mixture of sulphated galactans, which may also contain glucoronic acid or pyruvic acid. It dissolves in hot water and sets to gel on cooling. It is extensively used for culturing the microbes.
Chitin: Chitin forms the characteristic exoskeleton of invertebrates
like crab, lobster, prawns, etc. It is a polymer of acetylglucosamine.
Pectin: Pectins are present in apples, lemon and other fruits. In the
middle lamella of cell wall, pectin is found as calcium pectate. Pectins consist of repeating units of galactose and galacturonic acid.
Heteropolysaccharides: Let us learn more about the following
heteropolysaccharides:
Hemicellulose: It is found in association with cellulose in cell walls.
The commonly found sugars in hemicellulose are D-xylose, L-arabinose, D-galactose, and D-glucoronic acid.
Gums: Gums are substances exuded by plants on mechanical injury or
after bacterial, fungal or insect attack. The viscose substance helps to seal the wound and protects the plants. The polysaccharides in these are highly branched.
Hyaluronic acid: Hyaluronic acid is the simplest mucopolysaccharide
and linear polymer of disaccharides which form repeating units. Each disaccharide is linked to the next byÿb-1, 4-glucosidic bonds. It consists of two monosaccharides D-glucoronic acid and N-acetyl-D-glucosamine. It is found in the skin, vitreous humour of the eye, the umbilical cord, as a coating around ovum and in certain bacteria.
Heparin: Heparin is an anti-coagulant secreted by most cells in the
intestinal mucosa, liver, lung, spleen and kidney. It is a polymer of glucoronic acid and N-acetylglucosamine.
Figure 2.29 b–1, 4 glycosidic linkage.
O O C O O C O O C O O
Chondroitin sulphates: These are predominant in cornea, cartilage,
tendons, skin, heart valves and saliva. The repeating unit is a disaccharide of glucoronic acid linked to sulphite ester of N-acetylgalactosamine through a b-1,3-glycosidic bond.