UNIT 10 DNA - THE GENETIC MATERIAL Structure 10.1 Introduction Object~ves 10.2 Discovery of DNA as the Hereditary or Genetic Material 10.3 Nature of DNA - the Genetic Material DNA DNA Molecule - The Double Helix 10.4 DNA Replication DNA Replication is Semiconservative Mechanism of Replication Proofreading during Repliction DNA Replication is Remarkably Accurate 10.5 Genotype to Phenotype - Gene Expression DNA to Protein The Genet~cCode The Central Dogma Ribonucleic Acid (RNA) 10.6 Transcription Transcription in Prokaryotes Transcription in Eukaryotes RNA Polymerase Enzymes in Eukaryotes hn RNA or pre m RNA to m RNA 10.7 Translation Formation of mRNA Ribosome Complex and Chain Initiation Chain Elongation Chain Termination 10.8 Regulation of Gene Expression in Prokaryotes and Eukaryotes Housekeeping Genes, Inducible qenes and Repressible Genes Gene Express~onRegulation in Prokaryotes Gene Regulation I? Eukaryotes 10.9 Nuclear Basis of Differentiation and Development Totlpotency of a Differentiated Cell Differentiation and Development Result from Preprogrammed Circuits of Gene Expression Levels of Regulat~onof Gene Expression during Development Developmental Genetics in Certa~nModel Organisms Homeotic Genes and Homeoboxes 10.10 Oncogenes Cancer Cell Genetic Control of Cell Division Proto Oncogenes and Oncogenes The V~ralConnection of Oncogenes Poss~bleMechanism of Act~onof Oncogenes Tumour Suppressor Genes or Antioncogenes 10.1 1 Summary 10.12 Terminal Questions 10.1 INTRODUCTION - -- In the previous lessons you have learnt how life continues from generation to generation through the transfer of genes from parents to offspring. Thus through heredity and variation every organism is able to i) maintain species specificity, that is, look like other members of the species tb which.the organism belongs; ii) show resemblances with parents and relatives and also; iii) retain individual identity. This unit deals with the study of DNA, the molecule which constitutes the genes, its molecular structure and functioning. The unit will also provide information on cancer causing genes. jectives DNA - The Genetic iP iP Material F/fer studying this lesson you will be able to: Justify that DNA is the genetic material and describe the physical and chemical structure of DNA, List the enzymes and nucleotides required for DNA replication and describe the various steps of replication, Explain and illustrate the central dogma, Describe the structure of RNA and differentiate between the various types of RNA and explain the role of various types of RNA in protein synthesis, Describe the various steps of transcription and differentiate between transcription in prokaryotes and in eukaryotes, Explain the meanings of the terms constitutive, induction and repression and describe how genes are switched on and switched off, Explain the role of genes in development and differentiation, ldentify the role of oncogenes in causing cancer. itUdyguide Before taking up a detailed study of the structure and function of DNA, keep in mind the following: In a cell, chromosomes bear the genes in which hereditary information is stored. Prokaryotes have only one chromosome, whereas eukaryotes have several pairs of chromosomes. The number of chromosomes varies but is specific for a particular species and eukaryotic chromosomes are within the nucleus of each cell. The information for development and differentiation of a zygote is in its genes. The overall structure and function of the various kinds of cells of a multicellular organism or the single cell of unicellular organisms are dependent on the information present in the genes and the expression of those genes. Genes are segments of DNA. One chromosome has only one molecule of DNA. 0.2 DISCOVERY OF DNA AS THE HEREDITARY OR GENETIC MATERIAL the early twentieth century, scientists knew that chromosomes contain genes, the rs of hereditary information. Also they knew that chromosomes are made of and proteins but it took the first fifty years of the twentieth century and many ientists to discover that DNA is the genetic material. The evidence for this is briefly mmarized below: . Fredrich Miescher in 1898 isolated an acidic chemical from nuclei of pus cells 1 11 which he termed nuclein. Griffith's experiments demonstrated bacterial transformation. (Fig. 10.1). The bacterium Streptococcus pneumoniae was cultured in the laboratory on a nutrient agar medium and formed smooth colonies (S). These bacteria were injected into a mouse and the mouse died. A mutant of this bacterium forms rough colonies (R) because of the polysaccharide coat covering the bacterial cell and is not lethal to mice. In 1928, Frederick Griffith found that the rough nonvirulent bacteria get transformed into smooth virulent ones when mixed with heat killed virulent form. This phenomenon was termed bacterial transformation. ~bntihuit~OT Life r 1 a S sbain is avapsrlated and vWent nGsedcr Ma6elii Ma6e llves d Bloodsm@ehan dBadmuse- MauncLics IkRvinlentSBah Fig. 10.1: Griffith's bacterial transformation experiment. 3. In 1944, Oswald T. Avery, Colin M. Mcleod and Maclyn McCarty extracted DNA from the virulent Streptococcus pneumoniae after removing any protein or RNA present in the extract by protease and RNAse enzymes. (Fig. 10.2) This DNA extract when mixed with non-virulent strain of the bacteria transformed them into the virulent form. But when DNAse was added to the extract, no transformation took place. This was experimental proof of DNA being the transforming principle as the transformed bacteria divide and subsequent generations remain virulent and have a smooth coat. Avery and his colleagues, subjected the transforming principle to dircct physical and chemical analysis and found that it has characteristics exactly matching those of DNA. For example ultracentrifugation suggested that transforming principle was of very high molecular weight - characteristic of DNA. Electrophoresis showed that it has relatively high mobility, also characteristic of DNA. When placed in the spectrophotometer, the transforming principle absorbed uv light maximally of wave length of 260 nm. The absorption maxima of DNA is 260 nm. DNA - The Genetic -- Material ................ spun b ... :... bottom of tube -'=-w-% IIIScelk in mid&-pobehs liquid culture medium I Tmt with Treat with Treat rdlh he=f-mth Tmleomatb OCCUm OCOUrs -OrmL -OOQlt *cells+ IIISdk CQduskn.. cencwcs aftsveffxtor retlvefsdor b DNA b nat RNA 10.2: Avery, MacLeod and McCarty's experiment demonstrating that DNA is the transforming principle. IIIS=Smooth colonies, IIR=Rough colonies The virus which infect This experiment by Avery, Mcleod, McCarty suggested that DNA is the bacteria are termed hereditary material. bacteriophage. Further proof of DNA being the hereditary material came from experiments by Hershey and Chase. Alfred D. Hershey and Martha Chase (1952) used the TZ bacteriophage for their experiment. T2 viruses have a protein coat enveloping a DNA molecule. They labelled the phage protein coat with s3',a radioactive isotope of sulphur which would be incorporated into sulphur containing amino acids in the protein coat but not into DNA. When s~~labelled T2phage was nto bacteria, no label was detected in the new phage particles. On the d, when the phage was first labelled with P~~and then injected into the host bacteria, P~~which had got incorporated into phage DNA was nside the bacteria. ) Thus irbecame clear that the new generations of the bacteriophage inside the ; bacteria owed their existence to the phage DNA. This was proof enough that I DNA is the hereditary material. (Fig. 10.3 a and 10.3 b) Continuity of Life Vird DNA Wed Vidbrtl.wcd DNA labeled Prokin coat with "P ldbded wim 35s vilu5c?s harrlnfgted baderia Blends separates viral coats fmm bacteria a within bacterh Wnviral arats vi-p replication 00CU15 b c Fig.lO.3: Experiment of Hershey and Chase to demonstrate DNA as the hereditary material a) T2 Virus entering E.Coli, b) radioactive DNA inside bacteria, c) radioactive protein - outside bacteria. 5. Apart from experimental evidence, biochemical evidence due to certain facts such as those given below confirmed that DNA is the genetic material. i) Amount of DNA in every cell of an organism is the same. ii) Amount of DNA in gametes is half that of DNA present in somatic cells. iii) Amount of DNA is the same in all members of the same species. iv) Amount of DNA in a cell is constant and does not change by internal or external environment. v) DNA or amount of genetic information is proportional to evolutionary complexity. 10.3 NATURE OF DNA - THE GENETIC MATERIAL 10.3.1 DNA DNA or Deoxyribonucleic acid is a stable macromolecule. It is a polynucleotide. In other words, DNA is a polymer of monomeric units called deoxyribonucleotides. Each nucleotide consists of three subunits. i) a nitrogen containing heterocyclic aromatic ring - nitrogenous base ii) a pentose sugar - the deoxyribose sugar iii) a phosphate group (PO4) positioned on the sugar Nitrogenous base in a nucleotide may be a two-ring (bicyclic) base, either Adenine or Guanine called purine bases or a single-ring (monocyclic) base, either Thymine or Cytosine called pyrimidine bases. DNA - The Genetic The deoxyribose sugar is a 5-Carbon or pentose sugar (note positions C,, Cz, C3, C4, Material C5 in the figure 10.4). 1I 1 The combination of a base and a sugar is a nucleoside. Hence there are four I I deoxyribo nucleosides Adenine + deoxyribose sugar = deoxyadenosine Guanine + deoxyribose sugar = deoxyguanosine Thymine + deoxyribose sugar = deoxythymidine Cytosine + deoxyribose sugar = deoxycytidine A deoxynucleotide is deoxyribose sugar + nitrogenous base + phosphate. There are I I four nucleotides in DNA Adenine + deoxyribose sugar + phosphate Thynline + deoxyribose sugar + phosphate Guanine + deoxyribose sugar + phosphate Cytosine + deoxyribose sugar + phosphate II (Bas? + Sugar = Nu~kosie) (Base + Sugar + Phosphate = NuckoWe) Il Fig. 10.4: Components of a nucleoside and a nucleotide. 1 ( Chargaff's rule The four nucleotides (See figure 3.14 of Unit 3) are not present in equal amounts in a DNA molecule. The amounts vary widely among organisms.