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DNA as a Genetic Material

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DNA as a Genetic Material

  1. 1.  The genetic material must meet 4 criteria 1. Information 2. Transmission 3. Replication 4. Variation IDENTIFICATION OF DNA AS THE GENETIC MATERIAL
  2. 2. • By the early 1900’s it was known that the chromosomes carry the genetic (hereditary) information • Chromosomes consist of DNA (deoxyribonucleic acid) and proteins
  3. 3. 1909 1911 1950194419291865 Gregor Mendel: Introduces the concept of heredity
  4. 4. 1909 1911 1950194419291865 Wilhelm Johannsen: Coins the term “Gene
  5. 5. 1909 1911 1950194419291865 Thomas Hunt Morgan: Discovers that genes are responsible for inheritance
  6. 6.  Two strains of Streptococcus pneumoniae  S  Smooth ○ Secrete a polysaccharide capsule ○ Produce smooth colonies on solid media ○ virulent  R  Rough ○ Unable to secrete a capsule ○ Produce colonies with a rough appearance ○ avirulent  Two coat types  II  III  Four possible phenotypes  IIS or IIIS or IIR or IIIR 1928 - Frederick Griffith’s Transformation Experiments
  7. 7. Griffith’s Experiments 9-6  Rare mutations convert strains  S into R strain & vice versa  But mutations do not change coat types  II into III or vice versa
  8. 8. 9-8
  9. 9. 9-9  Something from the dead type IIIS transformed type IIR into type IIIS  Called this process transformation  The unknown substance was termed the transforming principle Griffith’s Conclusions
  10. 10. 1909 1911 1950194419291865 Phoebus Levene: Discovers that DNA is made up of nucleotides, phosphates, sugars and 4 bases
  11. 11. 1909 1911 1950194419291865 Oswald Avery: Shows that DNA can transform the property of cells However, this idea was not universally accepted
  12. 12.  Griffith’s transforming principle was the genetic material  Transformation assay to identify actual biomolecule  Major constituents - DNA, RNA, proteins, carbohydrates, & lipids  Made cell extracts from type IIIS cells containing each of these macromolecules 1944 - Avery, MacLeod & McCarty Identify the Genetic Material
  13. 13.  Mixed each extract with type IIR cells to test for transformation  Only extract containing purified DNA transformed type IIR to type IIIS  Verify that DNA, not RNA or protein, is the genetic material Avery’s Experiments
  14. 14. 1909 1911 1950194419291865 Erwin Chargaff: Shows that: A + G = T + C = 50%
  15. 15. • Used data of Erwin Chargaff, 1940’s and early 50's • Chargaff’s Rule: His data showed that in each species, the percent of A equals the percent of T, and the percent of G equals the percent of C. • Watson was shown this picture by Wilkins in early 1953. From the picture it was possible to calculate: 1) the distance between bases (3.4A) 2) the length of the period (34A) 3) the rise of the helix (36 degrees)
  16. 16. Franklin’s Work In 1951 Rosalind Franklin discovers the Two Forms of DNA through her X-ray diffraction. A – Dry Form B – Wet Form
  17. 17. X-Ray Crystallography
  18. 18. 1952- Hershey & Chase Confirm DNA is Genetic Material Using Phage T2
  19. 19. Life cycle of phage T2
  20. 20. Phage Attached to E. coli Cell
  21. 21. The Hershey and Chase experiment:  Used radioisotopes to distinguish DNA from proteins  32P labels DNA specifically  35S labels protein specifically  Infect non-radioactive E. coli with radioactively- labeled phages  Remove phage coats from cells  Is 32P or 35S inside bacteria?
  22. 22. Hershey & Chase Experiment S
  23. 23. Hershey & Chase Experiment
  24. 24. The Data 9-18
  25. 25.  In 1956, A. Gierer and G. Schramm isolated RNA from the tobacco mosaic virus (TMV)  Purified TMV RNA caused the same lesions as intact TMV viruses  Therefore, the viral genome is composed of RNA  Since that time, many RNA viruses have been found RNA is Genetic Material in Some Viruses
  26. 26. DNA Structure, Functions and Properties
  27. 27. DNA •DNA - a polymer of deoxyribo nucleotides •found in chromosomes, mitochondria and chloroplasts • carries the genetic information 28
  28. 28. Components of a nucleotide Base Sugar Phosphate
  29. 29. Nucleotide Nucleoside Base Phosphate Sugar X=H: DNA X=OH: RNA
  30. 30. Basic structure of pyrimidine and purine
  31. 31. Pyrimidines 32
  32. 32. Purines
  33. 33. Nomenclature of Nucleic Acid Components Base Nucleoside Nucleotide Nucleic acid Purines Adenine Adenosine Adenylate RNA Deoxyadenosine Deoxyadenylate DNA Guanine Guanosine Guanylate RNA Deoxy guanosine Deoxyguanylate DNA Pyrimidines Cytosine Cytidine Cytidylate RNA Deoxycytidine Deoxycytidylate DNA Thymine Thymidine Thymidylate DNA (deoxythymidine) (deoxythymidylate) Uracil Uridine Uridylate RNA
  34. 34. 5-Fluorouracil 6-Mercaptopurine Anticancer agents Azidothymidine Dideoxyinosine Antiretroviral agents Nucleoside and base analogs can be used as anti-cancer and anti-virus drugs
  35. 35. The primary structure of DNA is the sequence 5’ end 3’ end 5’ 3’ Phosphodiester linkage
  36. 36. Traditionally, a DNA sequence is drawn from 5’ to 3’ end. A shorthand notation for this sequence is ACGTA
  37. 37. The secondary structure of DNA is the double helix
  38. 38. The secondary structure of DNA Two anti-parallel polynucleotide chains wound around the same axis. Sugar-phosphate chains wrap around the periphery. Bases (A, T, C and G) occupy the core, forming complementary A · T and G · C Watson-Crick base pairs.
  39. 39.  hydrogen bonding;  base stacking The DNA double helix is held together mainly by- Hydrogen bonds
  40. 40. Hydrogen bond a chemical bond in which a hydrogen atom of one molecule is attracted to an electronegative atom, especially a nitrogen, oxygen, or fluorine atom, usually of another molecule.
  41. 41. Two hydrogen bonds between A:T pairs Three hydrogen bonds between C: G paired
  42. 42. Base Stacking The bases in DNA are planar and have a tendency to "stack". Major stacking forces: hydrophobic interaction van der Waals forces.
  43. 43. Variations in form of DNA  Most of the DNA - B-DNA or B-form DNA.  In certain condition, different forms of DNAs are found  A-DNA,Z-DNA,C-DNA,D-DNA,E-DNA.  This deviation in forms are based on their structural diversity.
  44. 44. Variations in form of DNA  B-DNA: Most common ,originally deduced from X-ray diffraction of sodium salt of DNA fibres at 92% relative humidity.  A-DNA: Originally identified by X-ray diffraction of analysis of DNA fibres at 75% relative humidity  Z-DNA: Left handed double helical structure winds to the left in a zig- zag pattern .
  45. 45.  C-DNA: Formed at 66% relative humidity and in presence of Li+ and Mg2+ ions.  D-DNA: Rare variant with 8 base pairs per helical turn ,form in structure devoid of guanine .  E- DNA: Extended or eccentric DNA.
  46. 46. 47
  47. 47. B-DNA  Described by James D. Watson & Francis crick.  Commonly found in DNA.  DNA molecule consists of 2 helical polynucleotide chains coiled around common axis.  2 helices are wound in such a way so as to produce 2 interchain spacing or groove –  Major/wide groove(width 12A°,depth 8.5A°)  Minor /narrow groove(width 6A°,depth 7.5A°)  These grooves provide surface with which proteins,chemicals,drugs can interact.
  48. 48.  2 helical wind along the molecules  2 chains run in opposite direction, they are antiparallel, the plane of bases are perpendicular to helix axis.  Right handed twisting  Uniform diameter (20A°)  Complementary base pairing  Base pair per turn is 10.4  Rise per base pair is 3.4A°
  49. 49. A-DNA  A-DNA is one of the possible double helical structure which DNA can adopt along with other two biologically active helix structure(B-DNA,Z-DNA).  Right handed double helix .  Short and fatty compared to B-DNA.  Occur only in dehydrated sample of DNA ,Such those used in crystallographic experiments.
  50. 50.  A-DNA was originally identified by X-ray diffraction analysis of DNA fibres at 75% relative humidity.  The grooves are not as deep in B-DNA.  The bases are more tilted  The base pairs per turn is 11.  Rise per base pair is 2.3A°.
  51. 51. Z-DNA  Left handed double helix structure winds to left in zig-zag manner.(DNA backbone were in zig-zag manner)so they are termed as Z-DNA.  Discovered by Rich, Nordheim &Wang in 1984.  It has antiparallel strands as B-DNA.  It is long and thin as compared to B- DNA.
  52. 52.  adjacent sugar have alternating orientation (against B-DNA which has same orientation).  Purines: syn confirmation (bases & sugar are near & on same side)  pyramidines: anti (bases & sugar are distant, on opposite sides)  Only one deep helical grooves.  There are 12 base pairs per turn with axial rise 3.8A° & angle of twist 60°.
  53. 53. C-DNA  C-DNA formed at 66% relative humidity (low)in presence of Li+ or Mg2+.  Right handed ,with axial rise of 3.32A° per base pair .  9.33 base pairs per turn.  Helical pitch 3.32A°×9.33°A=30.97A°.  Base pair rotation=38.58°.  Has diameter of19A°,smaller than that of A-&B-DNA.  The tilt of base is 7.8°
  54. 54. D-DNA  Extremely rare variant with only 8base pairs per helical turn .  This forms of DNA found in some DNA molecules devoid of guanine.  Axial rise of 3.03A°per base pairs .Tilt of 16.7° from axis of helix.  Actually 2 different forms of D-DNA 1. D(A):Takes part in D-A-B transition. 2. D(B):Associated with D-B change of confirmation.  2 DNA structure have same helical parameters.
  55. 55. Structure devoid of guanine
  56. 56. E-DNA  Cytosine methylation of or bromination of DNA sequence d(GGCGCC)2 is to induce a novel extended &eccentric double helix, which we call E-DNA.  E-DNA has a long helical axis rise and base perpendicular to the helical axis.  Deep major groove and shallow minor groove.  E-DNA allowed to crystallize for a period time longer, the methylated sequence forms standard A-DNA.
  57. 57.  E-DNA is the intermediate in the transition toA-DNA.  E-DNA is the intermediate in the crystallographic pathway from B-DNA to A-DNA.
  58. 58. CHARACTRISTICS A-DNA B-DNA C-DNA Z-DNA CONDITIONS 75% RELATIVE HUMIDITY;NA+,K +,Cs IONS 92% R.H:;LOW ION STRENGTH 60%R.H;Li/Mg IONS VERY HIGH SALT CONC. SHAPE BROADEST INTERMEDIATE NARROW NARROWEST HELIX SENSE RIGHT RIGHT RIGHT LEFT HELIX DIAMETER 25.5A° 20.7A° 19.0A° 18.4A° RISE PER BASE PAIR(H) 2.3A° 3.4A° 3.32A° 3.8A° BASE PAIR PER TURN(N) 11 10.4 9.33 12 HELIX PITCH(H×N) 25.5A° 35.36A° 30.97A° 45.60A° ROTATION PER BASE PAIR +32.72° +34.61° +38.58° -60° BASE PAIR TILT 19° 1° 7.8° 9° GLYCOSIDIC BOND ANTI ANTI _ ANTI FOR C,T. SYN FOR A,G. MAJOR GROOVE NARROW &VERY DEEP WIDE & QUITE DEEP _ NO MINOR GROOVE VERY BROAD & SHALLOW NARROW & QUITE DEEP _ VERY NARROW & DEEP
  59. 59. Does DNA fit the requirements of a hereditary material? Structure REQUIREMENT DNA Component Has biologically useful information to make protein Genetic code: 3 bases code for 1 amino acid(protein) Must reproduce faithfully and transmit to offspring Complementary bases are faithful: found in germ cells Must be stable within a living organism Backbone is strong covalent : hydrogen bonds Must be capable of incorporating stable changes Bases can change through known mechanisms
  60. 60. In eukaryotic cells, DNA is folded into chromatin
  61. 61. DNA Tertiary Structure •DNA DOUBLE HELICAL STRUCTURE COILS ROUND HISTONES. •DNA BOUND TO HISTONES FORMS NUCLEOSOMES (10nm FIBRES) •NUCLEOSOMES CONTAIN 146 NUCLEOTIDES
  62. 62. Nucleosomes any of the repeating globular subunits of chromatin that consist of a complex of DNA and histone
  63. 63. Structure of nucleosome core 68Biochemistry for Medics
  64. 64. Compaction of DNA in a eukaryotic chromosome 69Biochemistry for Medics
  65. 65. Supercoil = coil over coil 70Biochemistry for Medics
  66. 66. DNA melting and annealing 71Biochemistry for Medics
  67. 67. Reversible denaturation and annealing of DNA 72Biochemistry for Medics
  68. 68. Melting point (tm) of DNA The temperature at the mid-point of the transition 73Biochemistry for Medics
  69. 69. The tm of DNA depends on:  size of DNA  pH  ionic strength  Content of G·C base pairs 74Biochemistry for Medics
  70. 70. 75Biochemistry for Medics Functions of DNA and summary of structure DNA consists of four bases—A, G, C, and T—that are held in linear array by phosphodiester bonds through the 3' and 5' positions of adjacent deoxyribose moieties. DNA is organized into two strands by the pairing of bases A to T and G to C on complementary strands. These strands form a double helix around a central axis. The 3 x 109 base pairs of DNA in humans are organized into the haploid complement of 23 chromosomes. DNA provides a template for its own replication and thus maintenance of the genotype and for the transcription of the roughly 30,000 human genes into a variety of RNA molecules.

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