chapter 10 Genetics 445 There is enough experimental evidence t xcept in certain vi hat DNA i the genetic organisms excep 'ruses where RNA is the genetic material declared aS genetic material, attention was focused on chemical natur, knowledge on molecular level helped understanding the working of gene. CHEMICAL NATURE AND STRUCTURE OF DNA Hydrolysis of nucleic acid DNA showed that it is composed of: Phosphoric acid (H3PO3), a phosphate group. A5-C sugar, as deoxyribose sugar. terial in all living Once ONA was of ONA This so The purine bases, adenine (A) and guanine (6). 4. The pyrimidine bases, cytosine (C), and thymine (T), Both purine and pyrimidine bases contain significant amount of nitrogen, ther these are called nitrogenous bases. DNA is a Polymer DNA is a polymer. A polymer is a long chain-like molecule, comprising of numerous individual units, called (monomers, linked together in a series. The monomer in DNA molecule isanucleotide, Nucleotides —Fhe Monomers in DNA Chemical analysis showed that nucleic acid DNAis constructed of repeating units Called nucleotides. Nucleotides are found in the cell either as components of nucleic acids or “s nvidual molecules. Each nucleotide is composed of a sugar, a nitrogenous base, either ® purine or @ pyrimidine, and a phosphate molecule. The nitrogen base is linked to sugar. Amolecule comprising of sugar joined to a base is called a nucleoside. ‘gar Component of DNA 'n DNA the sugar component of the nucleotide is a pentose sugar, deoxyribose. It tin straight chain or ring form. It occurs in ring form in the nucleotide It contains a SY! group (-OH) attached to carbon atom number 2 witha hydragen group (-H) ‘ore ‘ The Su Cex Hyak 8 Ni Nitrogenous Bases These are complex « |146 Caravan's Textbook of Botany. Double-ring purines: the adenine and guanine; attached to the 1-catbon of the Sug, In DNA any one of these four different bases can be attached to this position The Phosphoric Acid Component group to the 5: earbon of the sugar © Upto three individual phosphate groups may be attaci “in series giving a nucleoside monophosphate (NMP), Ducleoside_ diphosphate | (NDP), nucleoside triphosphate (NTP) Nucleotide Nucleoside ae ie [AT |. | / Haworth structure inte tr hota a ribose | Hq {c) Phosphate groups _Cytosine (b) The nitrogenous basesa Gonotics 117 The Nomenclature of Nucleotides The naming of nucleotides depends upon the kind of nitrogenous base they contain, The four different nucleotides that polymerize to form DNA are: Pp Deoxyadenosine monophosphate or di Deoxyguanosine monophosphate or dGMP_ ye Deoxycytidine monophosphate or dCMP_ Panel a Deoxythymidine monophosphate or dTMP. ele They are also abbreviated as_A, G, C and T, when writing out the sequence of nucleotides found in a particular DNA molecule. The nucleotides exist as triphosphates in free cell condition, for example as_dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), d¢MP. (deoxyeytidine trjohosphate), and dTTP {deoxythymidine triphosphate). However{ before linkin 1m a polynucleotide, two phosphate molecules cleaye got, The one thal is left is called alpha- phospaate. 4 . Purine nucleotides. NH, ° Adenine Guanine on i | —p— | 0 OHH ‘Adenosine monophosphate. Pyrimidine nucleotides to y Cosine OH H OHH ‘Thymidine monophosphate Cytidine monophosphate110 Varavan & 1exuruun we on Polynucleotides The individual nucleotides are linked together to form a polymer calley polynucleotide. It is formed by attaching one nucleotide to another by way of the phosphat, groups A nucleotide of one kind need not to be followed by any particular other nucleotide. Nucleotides are Joined by Phosphodiester Bonds The nucleotide monomers are jgined by a phosphodiester bond: “phosphy indicating @ phosphorus atom and {diester referring to/two ester (C-O-P) bonds.'in each linkage. The phosphate group attached to the 5-carbon of one nucleotide is joined to 3. carbon of the next nucleotide. The bond can also be called 3-5 phosphodiester bond. Polynucleotides have Chemically Distinct Ends The two ends of the polynucleotide are not the same. One end is called 5’ or 5. terminus The 5-carbon at this end does not participate in a phosphodiester bond and 3 tnphosphate group is attached to it The other end of the molecule is called 3' or 3-O; terminus. At this the unreacted group is hydroxyl group. Polynucleotide has a Direction The chemical distinction between the two ends means that polynucleotides have ; direction, which can be looked as 5 '+3' (downward) or 3'— 5' (upward). PHY: UCTURE OF DNA By early 1950's a great deal was known about chemistry of DNA, i.e, it is compose: of nucleotides and these links together by phosphodiester bonds to form a polynuclectid: chain. Linus Pauling (1953), who had discovered alpha-helical structure of proteins propose: that DNA 1s a three stranded structure. But Watson and Crick differed and suggested a two-stranded structure for DN& molecule. Three lines, the chemical nature of the components of DNA, Chargaff's base ratios ‘and X-ray diffraction analysis, helped Watson and Crick to establish their model. Chargaff's Base Ratios Chargaff’s base ratios paved the way for the correct structure of DNA molecule Chargaff carried out a comprehensive analysis of the chemical composition of DNA. He usa new and sensitive (paper chromatographic tec! iques\to determine the exact amounts a nitrogenous bases in the samples of DNA purified from different tissues and differen organisms. The results revealed a(Gimple_mathematical_relationsh ip. that the number a adenine equals the number of thymines, and the number of guanines equals the number o cytosines A=Tand G= C. The total purines (A + G) will equal the total pyrimidines (T + C). ASL A summary of some of Chargaff's results indicating a consistent pattern to the ratios of the bases in DNA from different organismsGonotics 119 Ratio of bases in DNA samplos Organism Adenine: Thymine Guanine: Cytosine Cow 1.04 1.00 ‘ Man. 1,00 1.00 Salmon 1.02 1.02 Escherichia coli 1.09 0.99 Percentage Base Composition of Some DNAs Species Adenine Thymine Guanine = Cytosine Human Being (Liver) 30.3 30.3 19.5 19.9 Mycobacterium tuberculosis 15.1 14,'5 34.9 35.4 Sea Urchin 32.8 32.1 17.7 18.4 X-ray Diffraction Analysis X-ray diffraction analysis indicates that DNA #of evidence available to Watson and Crick was the X-ray diffract a crystalized DNA fibre is bombarded with X-rays. The theory behind X-rays diffraction analysis is based on the fact that the angles at which X-rays are deflected on passage through a crystal will be determined by the three- dimensional structure of the molecule in the crystal. The molecules deflections can be recorded by allowing the X-ray beam, after passage through the crystal, to expose to a photographic film. The result is a pattern of spots, the position and intensities of which may allow the structure of the molecule to be deduced. Maruice Wilkins, but (Rosalind Franklin)obtained clear pictures of DNA crystals and showed that DNA is a helix with two regular periodicities of 3.4 “A f 3.4 A and 34 A along the axis of the axis of the molecule. WATSON & CRICK MODE! Watson and Crick utilized X-ray diffraction paterns and information from chemical analysis (Chargaff's base ratios) to construct a scale mode! for DNA molecule. The important features of Watson and Crick model are: i. Double Helix Nature of DNA Molecule The diffraction patterns suggested that DNA molecule is not composed of a single polynucleotide but of two polynucleotides. These polynucleotide chains are twisted. in a spiral. Therefore, the DNA molecule is in the form of a double helix. molecule. The second piece ion pattern obtained whenGee aa 120 Caravan's Textbook of Botany us Bases and Right-handed Double Helix 7 ugar-phosphate molecules form backbone of the polynucleotide ch; [esembing the tailing of a staircage.)The base pairs extend from the sugar-phosph ‘backbone and are stacker inside the double hy bling the stairs of the sp staircase. The double hel executes a tum every\ten base pairs! The pitch of the heli 34_A) meaning that the spacing between adjacent base pairing is{3.4°A) Vv Nitrogenous bases Sugar phosphate backbone Hydrogen bonds: ‘© o@ =0 Cin phosphate Cand Ni eBametar ot ® ° iii, Base Pairing and Hydrogen Bonding The pairing was found to be preféréntial, the purine adenine (A) prefer to pair wit pyrimidine thymine (T), whereas the purine guanine (G) pair with pyrimidine cytosine (C). was observed! that hydrogen bonds hold the base-pairs together. Two hydrogen bonds hol A:T pair_and three G-C pair. The two polynucleotide chains are held together because 0 these hydrogen bonds. iv. Double Helix his Two Different Grooves The sugars in the chin are so arranged that it causes the double helix to contain two unequal grooves, one conspicuot Si roe eG other, re larger groove is called mejor groove.and the smaller minor groove.(This feature is important in fhe interaction between the double helix and the proteins involved in DNA replication and in the expression of genetié nema)a Gonetics _121 v. Antiparallel Arrangement and Polarity - The two polynucleotide chains of a DNA molecule are not identical. due to preferential pairing. The‘ chains run in opposite direction, for example if the orientation of one chain is 3'45', the other chain would be in reverse order and would run 5'-»3". As a result of this orientation, the molecule looks the same from the tops as well as from the bottom because a 3' chain and a 5' chain terminates at each end. Each strand exhibit polarity, i.e., one end of the strand has 5 phosphate and the other end a 3' phosphate group. Therefore, the two strands arefanti aa vi. Complementary The DNA strands show complementary, i.e., the A and C in one strand correspond to a T and G in the other. The two polynucleotides in a double helix are therefore complementary. ‘e., the sequence of one’ determining the sequence of the other. ALTERNATIVE FORMS OF DNA DNA exists in many forms, such as: i. BDNA (wilson extck ud Dye) D las3 The form of DNA described above is called B DNA. It j is, Sf sores gros It turns in clockwise manner when viewed down its axis. The bases are stacked aoe exactly perpendicular to the main axis with wetinnania ttt Ao bERA i, ADNACyied by deh gata. 4b If the igplzgcontents vdecce coout %6, the A fern gray will ocguF.I9 this form the bases aré tied with regard ip the as ‘land there are mére base pair the Sef tut as” compared to B DNA. Hoye ann Eom PA— 77 « iii, ZDNA It is a left-handed helix. The sugar-| eppaprate backbone forms a zigzag structure. The Z DNA looks like B DNA in which each Bae was rotated 180 degrees, resulting in a Zigzag left-handed 38 ot Z PNA js thoyght to bev invalv equiating gen Gagared becouse. 6f TR ten rabsce - seston ne destruction - ‘vgn oat a) DNA DENATURATION moe 1 bono a* more empl The hydrogen bonds between the base pairs are weak individually but because these are present in large number in a DNA molecule,therefore they provide structural stability to the DNA molecule. These bonds can be broken and the DNA strands can be separated by beating the DNA molecule. The hydrogen bonds denature or melt at a specific temperature. It has beén observed that more the hydrogen bonds in a DNA molecule, the higher the temperature needed to denature it. Because three hydrogen bonds hold the base pair G-C, therefore the higher the G-C contents in a DNA molecule, the higher the temperature is required to denature that DNA. DNA REPLI N ~ Before Watson and Crick suggested model for DNA, it was known that DNA undergoes (gelf-duplicationor_teplication) but there was no idea how it takes place.© Lon Sh syne —9 unwind DNA 122 _Caravan’s Textbook otpotan 2 nakes. Pepe M > Prov Sl = iy One of the mas ae ae an “fi model is inal it of a ave. C2 units replication. oly “ ase, “start seen ROH "y Wim Ci deotid fide Watson and Crick reese ll Primes proposed a possible mechanism of complementary replication based on sted model. strands of DNA Their observation i the property of ees According to them: a. The hydrogen bonds between the “bases of two complementary chains dissolve and the two chains % unwind DNA replicating a b. Each strand maintains its ae \ 7 integtity in the process and db \ io does not break down => 5 : building uns ©. Each stratd acts as a ch a template (a guide or _blue- rint) or pattern for assembly of another strand, one which is complementary to it. d. The complementary strands are constructed from the building material of the nucleotide present in the cell. © As the _sugar-phosphate sbackbone is assembled, each nitrogen base in the original —riew chain complementary strands strand attracts the complementary one. The adenine (A) in one chain attracts the Thymine (T). This complementary attraction by all the bases in each of the two original strands, along with the assembly of sugar-phosphate backbone, results in the construction of two new chains, each complementary to the original old ones. The enzyme[DNA palymerasd catalyze the process of replication, chemes for DNA Replication There are three possible schemes for DNA replication. These are:i. _ _Semiconservative Replication The above mentioned mechanism for DNA molecule replication , is, Ig semiconservative replication. because the entire double helix does not remain eae new DNA is being formed, instead ithe two strands come apart. However, each strand is preserved intact. Every daughter DNA molecule has an intact template strand and ardeteet newly replicated strand ii. Conservative me igation (0 “4, genved.) Itis alternative method of repficatioh. In this type of replication the original double helix stay together and a new double stranded molecule is built up next to them. The original double helix-acts as template for a new one, one daughter molecule would consist of the original parent DNA and the other daughter would be totally new DNA. Dispersive Replication In this type of replication the original strands break down and entirely new strands are constructed from these and other precursors in the cell. Some parts of original double helix are conserved and some parts are not. Daughter molecules would consist of part template and part newly synthesized DNA. a ___ Genetics 123 Parent molecule Spge F 3 Parent Daughter strand strand _—— a molecules ~~, kde", ene Fluentk Semi-conservative Conservative Dispersive124 _Caravan's Textbook of 8: Density-Gradient Centrifugation a In this technique {cesium chloride (CsC1) solutionis, spun in an ultracentrifuge, high speed for several hours. The solution is distributed from one end of the tube to the gj {twill eventually reach an equilibrium, and at this time, there is a density gradient in the 4, The cesium chloride molecules will be more dense at the centrifugal end (base of the |, and the density decreases gradually towards the centripetal end (top of the tube). If ON, any other substance) is added, it will concentrate and form a band in the tube at the» where its density is the same as that of cesium chloride. If there are several types of with different densities, they will form several bands. The bands can be detecteg observing the tubes with ultraviolet ight at a wavelength of 260 nm) in which nucleic a absorb strongly. ieee! |ESELSO! --- Experimental Evidence of Replication anreneeneerae Meselson and Stahl in 1958, provided a strong support for the concept of replica as suggested by Watson and Crick. They designed an experiment to determine the mos {8% DNA replication. They grew & col na medumcontaining a heavy isotope of nitrogen‘ ‘Dep (the normal form of nitrogen is “N). After growing for several generations on the =N medi 3 the DNA of E. coi becomes denser (heavy). They employed a technique called_den be gradient centrifugation to determine the density of the DNA strands. When extracted? pals, tis Dazapaetes out in the density gradient at a characteristics position. (4 Meselson and Stahl transferred the bacteria with beays SN) one ig @ med ty a | Ti) va, containing only “N. The time of transfer was taken as {ime = OY the'E: coil cells allowed to replicate during several generations with cell samples removed at ver intervals. From each sample, pit, was isolated and subjected to density-grai centrifugation OM *solacsion Q After one generation, the new DNA, replicated in “N medium, was intermedia 6 density between light ('4N) and heavy ('SN) DNA. Each molecule composed of one new strand and one old 15N strand. This suggests semiconservative replication. If replication 5 been conprvative, there would have been two bands at the first generation of replicatior original 'NI DNA and a new “'N double helix. Similarly if the method of replication hadt dispersive, the result would have been various multiple bands of DNA. After two cell divisions, DNA samples showed two density bands: one intermediate and the other lighter. Similar results occurred in third generation, howevel proportion of “N bands (light bands) increased. aa results of Meselson-Stahl experiment provide strong support for semicbhidarSive mode of f DNA replication as proposed by Watson and Cry Replication is Bidirectional Gains established that replication initiates at a single site and It is bidirectione. the two replicating sites move in opposite directions around the circular DNA. He determined the time of movement of this fork around the circular DNA. The forkGenetics 125 estimated ‘to travel approximately 20-30 u/min. Recent research evidences support Caim's observations. Replication in Eukaryotes a In eukaryotes the DNA molecules (chromosomes) are larger than in prokaryotes and are not circular. There are usually multiple sites of i replication. Thus each eukaryotic chromosome is composed of many replicating site called{replicons.} jin comparison to E. coli chromosome which is composed of only one replicon. In eukaryotes these replicating units form "bubble" or “eyes" in the DNA during replication. The second category of nucleic acids is the ribonucleic acid or RNA. The building biocks of RNA are also nucleotides linked into polynucleotide chains. However, it differs from DNA in the following respects: a. Ribose sugar replaces deoxyribose. Ribose differs from the deoxyribose sugar in that the deoxyribose has one less oxygen at C-2 position than does the ribose b. The pyrimidine base uracil replaces thymine. c. The polynucleotides that polymerize to make RNA are i. Adenosine monophosphate ii, Guanosine monophosphate ii. Cytidine monophosphate fii. Uridine monophosphate The individual nucleotides are linked together through 3' — 5' phosphodieste~ bonds. d. In most instances the RNA is single-stranded, linear molecule. However in some cases the molecules fold back on themselves, and in some animal viruses which have RNA as their genetic material it is in the form a double-stranded helix. Te