Tamilnadu Board Class 12 Zoology Chapter 5

The differential labelling thus enabled them toidentify DNA and proteins of the phage.Hershey and Chalse mixed the labelledphages with unlabeled E. coli and allowedbacteriophages to attack and inject theirgenetic material. Soon after infection (beforelysis of bacteria), the bacterial cells were gentlyagitated in a blender to loosen the adheringphase particles. It was observed that only 32Pwas found associated with bacterial cells and35S was in the surrounding medium and notin the bacterial cells. When phage progenywas studied for radioactivity, it was foundthat it carried only 32P and not 35S (Fig. 5.2).These results clearly indicate that only DNAand not protein coat entered the bacterialcells. Hershey and Chase thus conclusivelyproved that it was DNA, not protein, whichcarries the hereditary information from virusto bacteria.5.4 Chemistry of Nucleic AcidsHaving identified the genetic material asthe nucleic acid DNA (or RNA), we proceedto examine the chemical structure of thesemolecules. Generally nucleic acids are along chain or polymer of repeating subunitscalled nucleotides. Each nucleotide subunit iscomposed of three parts: a nitrogenous base,a five carbon sugar (pentose) and a phosphategroup.Pentose sugarThere are two types of nucleic acidsdepending on the type of pentose sugar.Those containing deoxyribose sugar are calledDeoxyribo Nucleic Acid (DNA) and thosewith ribose sugar are known as RibonucleicAcid (RNA). DNA is found in the nucleusof eukaryotes and nucleoid of prokaryotes.The only difference between these twosugars is that there is one oxygen atom less indeoxyribose.Molecular GeneticsNitrogenous basesThe bases are nitrogen containingmolecules having the chemical propertiesof a base (a substance that accepts H ion orproton in solution). DNA and RNA both havefour bases (two purines and two pyrimidines)in their nucleotide chain. Two of the bases,Adenine (A) and Guanine (G) have doublecarbon–nitrogen ring structures and arecalled purines. The bases, Thymine (T),Cytosine (C) and Uracil (U) have single ringstructure and these are called pyrimidines.Thymine is unique for DNA, while Uracil isunique for RNA.The phosphate functional groupIt is derived from phosphoric acid(H3PO4), has three active OH- groups of whichtwo are involved in strand formation. Thephosphate functional group (PO4) gives DNAand RNA the property of an acid (a substancethat releases an H ion or proton in solution)at physiological pH, hence the name nucleicacid. The bonds that are formed fromphosphates are esters. The oxygen atom of thephosphate group is negatively charged afterthe formation of the phosphodiester bonds.This negatively charged phosphate ensuresthe retention of nucleic acid within the cell ornuclear membrane.Nucleoside and nucleotideThe nitrogenous base is chemicallylinked to one molecule of sugar (at the1-carbon of the sugar) forming a nucleoside.When a phosphate group is attached to the 5'carbon of the same sugar, the nucleosidebecomes a nucleotide. The nucleotides arejoined (polymerized) by condensationreaction to form a polynucleotide chain. Thehydroxyl group on the 3' carbon of a sugar ofone nucleotide forms an ester with thephosphate of another nucleotide. Thechemical bonds that link the sugar72TN GOVT XII Zoology chapter5.indd 7225-02-2019 18:23:04

components of adjacent nucleotides are calledphosphodiester bond (5'3'), indicatingthe polarity of the strand.The ends of the DNA or RNA are distinct.The two ends are designated by the symbols 5'and 3'. The symbol 5' refers to carbon in thesugar to which a phosphate (PO4) functionalgroup is attached. The symbol 3' refers tocarbon in the sugar to which hydroxyl (OH)functional group is attached. In RNA, everynucleotide residue has an additional –OHgroup at 2' position in the ribose.Understanding the 5'3' direction of anucleic acid is critical for understanding theaspects of replication and transcription.Based on the X - ray diffraction analysis ofMaurice Wilkins and Rosalind Franklin, thedouble helix model for DNA was proposed byJames Watson and Francis Crick in 1953. Thehighlight was the base pairing between thetwo strands of the polynucleotide chain. Thisproposition was based on the observationsof Erwin Chargaff that Adenine pairs withThymine (A T) with two hydrogen bondsand Guanine pairs with Cytosine (G C)with three hydrogen bonds. The ratiosbetween Adenine with Thymine and Guaninewith Cytosine are constant and equal. Thebase pairing confers a unique property tothe polynucleotide chain. They are said to becomplementary to each other, that is, if thesequence of bases in one strand (template) isknown, then the sequence in the other strandcan be predicted. The salient features of DNAstructure has already been dealt in class XI.5.5 RNA worldA typical cell contains about ten times asmuch RNA as DNA. The high RNA contentis mainly due to the variety of roles played byRNA in the cell. Fraenkel-Conrat and Singer(1957) first demonstrated that RNA is thegenetic material in RNA containing viruseslike TMV (Tobacco Mosaic Virus) and theyseparated RNA from the protein of TMVviruses. Three molecular biologists in the early1980’s (Leslie Orgel, Francis Brick and CarlWoese) independently proposed the ‘RNAworld’ as the first stage in the evolution oflife, a stage when RNA catalysed all moleculesnecessary for survival and replication. Theterm ‘RNA world’ first used by Walter Gilbertin 1986, hypothesizes RNA as the first geneticmaterial on earth. There is now enoughevidence to suggest that essential life processes(such as metabolism, translation, splicing etc.,)evolved around RNA. RNA has the ability to actas both genetic material and catalyst. There areseveral biochemical reactions in living systemsthat are catalysed by RNA. This catalyticRNA is known as ribozyme. But, RNA beinga catalyst was reactive and hence unstable.This led to evolution of a more stable form ofDNA, with certain chemical modifications.Since DNA is a double stranded moleculehaving complementary strand, it has resistedchanges by evolving a process of repair. SomeRNA molecules function as gene regulators bybinding to DNA and affect gene expression.Some viruses use RNA as the genetic material.Andrew Fire and Craig Mellow (recipients ofNobel Prize in 2006) were of the opinion thatRNA is an active ingredient in the chemistry oflife. The types of RNA and their role have beendiscussed in class XI.5.6 Properties of genetic material(DNA versus RNA)The experiment by Hershey and Chaseclearly indicates that it is DNA that actsas a genetic material. However, in someviruses like Tobacco mosaic virus (TMV),bacteriophage θB, RNA acts as the geneticmaterial. A molecule that can act as a geneticmaterial should have the following properties: Self Replication: It should be able toreplicate. According to the rule of basepairing and complementarity, both nucleic73TN GOVT XII Zoology chapter5.indd 73Molecular Genetics25-02-2019 18:23:04

acids (DNA and RNA) have the ability todirect duplications. Proteins fail to fulfillthis criteria.a faster rate. Thus viruses having RNAgenome with shorter life span can mutateand evolve faster.Stability: It should be stable structurally andchemically. The genetic material should bestable enough not to change with differentstages of life cycle, age or with change inphysiology of the organism. Stability as oneof property of genetic material was clearlyevident in Griffith’s transforming principle.Heat which killed the bacteria did notdestroy some of the properties of geneticmaterial. In DNA the two strands beingcomplementary, if separated (denatured) byheating can come together (renaturation)when appropriate condition is provided.Further 2' OH group presentat every nucleotide in RNA is areactive group that makes RNAliable and easily degradable. RNAis also known to be catalytic andreactive. Hence, DNA is chemicallymore stable and chemically lessreactive when compared to RNA.Presence of thymine instead ofuracil in DNA confers additionalstability to DNA.The above discussion indicates that bothRNA and DNA can function as a geneticmaterial. DNA is more stable, and is preferredfor storage of genetic information.Information storage: It should beable to express itself in the formof ‘Mendelian characters’. RNAcan directly code for proteinsynthesis and can easily expressthe characters. DNA, howeverdepends on RNA for synthesisof proteins. Both DNA and RNAcan act as a genetic material, butDNA being more stable storesthe genetic information and RNAtransfers the genetic information.5.7 Packaging of DNA helixThe distance between two consecutivebase pairs is 0.34nm (0.34 10-9m) of theDNA double helix in a typical mammaliancell. When the total number of base pairs ismultiplied with the distance between twoconsecutive base pairs (6.6 10-9 0.34 10-9m/bp), the length of DNA double helix is 1XFOHRVRPH&RUH '1 % /LQNHU '1 % '&)( % DQG WZRPROHFXOHV HDFK '1 LVWRQH RFWDPHU LVWRQH SURWHLQVariation through mutation: It1XFOHRVRPHshould be able to mutate. BothDNA and RNA are able to mutate. Fig. 5.3 Condensation of DNA - A - DNA, B-NucleosomesRNA being unstable, mutates at and Histones, C- Chromatin fiber, D- Coiled chromatinfiber, E- Coiled coil, F- metaphase chromatidMolecular Genetics74TN GOVT XII Zoology chapter5.indd 7425-02-2019 18:23:05

approximately 2.2 m. (The total length of thedouble helical DNA total number of basepairs distance between two consecutivebase pairs). If the length of E. coli DNA is1.36 mm, the number of base pairs in E. coliis 4 106m (1.36 103 m/0.34 10-9). Thelength of the DNA double helix is far greaterthan the dimension of a typical mammaliannucleus (approximately 10-6 m). How is sucha long DNA polymer packaged in a cell?Chromosomes are carriers of genes whichare responsible for various characters fromgeneration to generation. Du Praw (1965)proposed a single stranded model (unineme),as a long coiled molecule which is associatedwith histone proteins in eukaryotes. Plantsand animals have more DNA than bacteriaand must fold this DNA to fit into the cellnucleus. In prokaryotes such as E. coli thoughthey do not have defined nucleus, the DNAis not scattered throughout the cell. DNA(being negatively charged) is held with someproteins (that have positive charges) in aregion called the nucleoid. The DNA as anucleoid is organized into large loops heldby protein. DNA of prokaryotes is almostcircular and lacks chromatin organization,hence termed genophore.In eukaryotes, this organization is much morecomplex. Chromatin is formed by a series ofrepeating units called nucleosomes. Kornbergproposed a model for the nucleosome, inwhich 2 molecules of the four histone proteinsH2A, H2B, H3 and H4 are organized toform a unit of eight molecules called histoneoctamere. The negatively charged DNAis wrapped around the positively chargedhistone octamere to form a structure callednucleosome. A typical nucleosome contains200 bp of DNA helix. The histone octameresare in close contact and DNA is coiled onthe outside of nucleosome. Neighbouringnucleosomes are connected by linker DNA(H1) that is exposed to enzymes. The DNAmakes two complete turns around the histoneoctameres and the two turns are sealed off byan H1 molecule. Chromatin lacking H1 has abeads-on-a-string appearance in which DNAenters and leaves the nucleosomes at randomplaces. H1 of one nucleosome can interactwith H1 of the neighbouring nucleosomesresulting in the further folding of the fibre.The chromatin fiber in interphase nuclei andmitotic chromosomes have a diameter thatvary between 200-300 nm and representsinactive chromatin. 30 nm fibre arises from thefolding of nucleosome, chains into a solenoidstructure having six nucleosomes per turn. Thisstructure is stabilized by interaction betweendifferent H1 molecules. DNA is a solenoid andpacked about 40 folds. The hierarchical natureof chromosome structure is illustrated in (Fig.5.3). Additional set of proteins are requiredfor packing of chromatin at higher level andare referred to as non-histone chromosomalproteins (NHC). In a typical nucleus, someregions of chromatin are loosely packed (lightlystained) and are referred to as euchromatin.The chromatin that is tightly packed (staineddarkly) is called heterochromatin. Euchromatinis transcriptionally active and heterochromatinis transcriptionally inactive.5.8 DNA ReplicationReplication of DNA takes place during theS phase of cell cycle. During replication, eachDNA molecule gives rise to two DNA strands,identical to each other as well as to the parentstrand. Three hypotheses of DNA replicationhave been proposed. They are conservativereplication, dispersive replication, and semiconservative replication.In conservative replication, the originaldouble helix serves as a template. The originalmolecule is preserved intact and an entirelynew double stranded molecule is synthesized.In dispersive replication, the original moleculeis broken into fragments and each fragment75TN GOVT XII Zoology chapter5.indd 75Molecular Genetics25-02-2019 18:23:05

5'3'T AC GA TParentC GC GG CT AC GT AC GT AT AATParentalstrandA3'CC GG CT AT A5'TParentalstrandCC GG CTAC GT AC GA TC GT AC GT AA TT AA T3'5.8.1 Experimental proof ofDNA oposed by Watson and Crick in 1953. Thismechanism of replication is based on theDNA model. They suggested that the twopolynucleotide strands of DNA moleculeunwind and start separating at one end.During this process, covalent hydrogen bondsare broken. The separated single strand thenacts as template for the synthesis of a newstrand. Subsequently, each daughter doublehelix carries one polynucleotide strand fromthe parent molecule that acts as a templateand the other strand is newly synthesisedand complementary to the parent strand(Fig. 5.4).DaughterstrandsFig. 5.4 Semiconservative DNA replicationserves as a template for the synthesis ofcomplementary fragments. Finally two newmolecules are formed which consist of bothold and new fragments.The mode of DNA replication wasdetermined in 1958 by Meselson and Stahl.They designed an experiment to distinguishbetween semi conservative, conservative anddispersive replications. In their experiment,they grew two cultures of E.coli for manygenerations in separate media. The ‘heavy’culture was grown in a medium in whichthe nitrogen source (NH4Cl) contained theheavy isotope 15N and the ‘light’ culture wasgrown in a medium in which the nitrogenGeneration I151514N-DNAGeneration IIN-DNA14N-DNA1520 min1440 min14Gravitational -DNAN-DNAN-DNANHybridFig. 5.5 Meselson and Stahl experiment to support semiconservative mode of DNAreplicationMolecular Genetics76TN GOVT XII Zoology chapter5.indd 7625-02-2019 18:23:05

source contained light isotope 14N for manygenerations. At the end of growth, theyobserved that the bacterial DNA in the heavyculture contained only 15N and in the lightculture only 14N. The heavy DNA could bedistinguished from light DNA (15N from 14N)with a technique called Cesium Chloride(CsCl) density gradient centrifugation. Inthis process, heavy and light DNA extractedfrom cells in the two cultures settled into twodistinct and separate bands (hybrid DNA)(Fig. 5.5).The heavy culture (15N) was then transferredinto a medium that had only NH4Cl, and tooksamples at various definite time intervals (20minutes duration). After the first replication,they extracted DNA and subjected it to densitygradient centrifugation. The DNA settledinto a band that was intermediate in positionbetween the previously determined heavy andlight bands. After the second replication (40minutes duration), they again extracted DNAsamples, and this time found the DNA settlinginto two bands, one at the light band positionand one at intermediate position. These resultsconfirm Watson and Crick’s semi conservativereplication hypothesis.errors are corrected by repair enzymes suchas nucleases. Deoxy nucleotide triphosphateacts as substrate and also provides energy forpolymerization reaction.Replication begins at the initiation site calledthe site of ‘origin of replication’ (ori). Inprokaryotes, there is only one origin ofreplication, whereas in eukaryotes with giantDNA molecules, there can be several originsof replication (replicons). Since the twostrands of DNA cannot be separatedthroughout at a time (due to large requirementof energy) the replication occurs within asmall opening of the DNA helix called asreplication fork. Unwinding of the DNAstrand is carried out by DNA helicase. Thus, inone strand (template strand with polarity3'5') the replication is continuous and isknown as the leading strand while in theother strand (coding strand with polarity5'3') replication is discontinuous, knownas the lagging strand (Fig. 5.6).Thediscontinuously synthesized fragments of thelagging strand (called the Okazaki fragments)are joined by the enzyme DNA ligase.5.8.2 Enzymes and mechanismof replicationIn prokaryotes, replication process requiresthree types of DNA polymerases (DNApolymerase I, II, and III). DNA polymeraseIII is the main enzyme involved in DNAreplication. DNA polymerase I (also knownas Kornberg enzyme) and DNA polymeraseII are involved in DNA repair mechanism.Eukaryotes have five types of DNApolymerases that catalyses the polymerizationof nucleotides at the 3' OH of the new strandwithin a short period of time. E.coli that has4.6 X 106 bp completes its replication processwithin 38 minutes. Replication takes placefaster at the same time accurately. Any errorwill lead to mutation. However replicationFig 5.6 Mechanism of replication showing areplication fork77TN GOVT XII Zoology chapter5.indd 77Molecular Genetics25-02-2019 18:23:05

As they move away in both directions, newlysynthesized complementary nucleotides arepaired with the existing nucleotides on theparent strand and covalently bonded togetherby DNA polymerase. Formation of new strandrequires a primer (a short stretch of RNA)forinitiation. The primer produces a 3'-OH end onthe sequence of ribonucleotides, to which deoxyribonucleotides are added. The RNA primer isultimately removed leaving a gap in the newlysynthesized DNA strand. It is removed from5' end one by one by the exonuclease activityof DNA polymerase. Finally, when all thenucleotides are in position, gaps are sealed bythe enzyme DNA ligase.At the point of origin of repliction, thehelicases and topoisomerases (DNA gyrase)unwind and pull apart the strands, forming aY-Shaped structure called the replication fork.There are two replication forks at each origin.The two strands of a DNA helix have anantiparallel orientation. The enzyme DNApolymerase can only catalyse the addition of anucleotide to the new strands in the 5'3'direction, as it can only add nucleotides to the3' carbon position.5.9 TranscriptionFrancis Crick proposed the Central dogmain molecular biology which states that geneticinformation flows as follows:The process of copying genetic informationfrom one strand of DNA into RNA is termedtranscription. This process takes place inpresence of DNA dependent RNA polymerase.In some retroviruses that contain RNA asthe genetic material (e.g, HIV), the flow ofMolecular Geneticsinformation is reversed. RNA synthesizes DNAby reverse transcription, then transcribed intomRNA by transcription and then into proteinsby translation.For a cell to operate, its genes must beexpressed. This means that the gene products,whether proteins or RNA molecules must bemade. The RNA that carries genetic informationencoding a protein from genes into the cell isknown as messenger RNA (mRNA). For a geneto be transcribed, the DNA which is a doublehelix must be pulled apart temporarily, andRNA is synthesized by RNA polymerase. Thisenzyme binds to DNA at the start of a gene andopens the double helix. Finally, RNA moleculeis synthesized. The nucleotide sequence in theRNA is complementary to the DNA templatestrand from which it is synthesized.Both the strands of DNA are not copied duringtranscription for two reasons. 1. If both the strandsact as a template, they would code for RNA withdifferent sequences. This in turn would code forproteins with different amino acid sequences.This would result in one segment of DNA codingfor two different proteins, hence complicate thegenetic information transfer machinery. 2. If twoRNA molecules were produced simultaneously,double stranded RNA complementary to eachother would be formed. This would prevent RNAfrom being translated into proteins.5.9.1 Transcription unit and geneA transcriptional unit in DNA is defined bythree regions, a promoter, the structural geneand a terminator. The promoter is locatedtowards the 5' end. It is a DNA sequence thatprovides binding site for RNA polymerase. Thepresence of promoter in a transcription unit,defines the template and coding strands. Theterminator region located towards the 3' endof the coding strand contains a DNA sequencethat causes the RNA polymerase to stoptranscribing. In eukaryotes the promoter hasAT rich regions called TATA box (Goldberg-78TN GOVT XII Zoology chapter5.indd 7825-02-2019 18:23:05

Fig. 5. 7 Schematic structure of a transcriptio

Hershey-Chase experiment finally provided convincing evidence that DNA is the genetic material. 5.3.1 Hershey and Chase experiment on T. 2. bacteriophage. Alfred Hershey. and . Martha Chase (1952) conducted experiments on bacteriophages that infect bacteria. Phage T. 2. is a