1.2 DNA Is the Genetic Material of Bacteria and Viruses

Transcription

1 1.1 Introduction chromosome A discrete unit of the genome carrying many genes.(hereditary information)-- -Sequence of DNA Each chromosome consists of a very long molecule of duplex DNA and an approximately equal mass of proteins, and is visible as a morphological entity only during cell division. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated.

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3 1.2 DNA Is the Genetic Material of Bacteria and Viruses

4 Isolate DNA first time in 1868 Friedrich Miescher (August 1844 August 1895) Isolated something no one had ever seen before from the nuclei of cells He called the compound nuclein which will be called DNA (deoxyribo-nucleicacid) and RNA (ribo-nucleic-acid)

5 Frederick Griffith Bacterial transformation provided the first support that DNA is the genetic material of bacteria.(frederick Griffith, 1928) S- Smooth: virulence, shinny colony R- Rough: avirulence, rough appearance. During transformation, genetic properties can be transferred from one bacterial strain to another by extracting DNA from the first strain and adding it to the second strain.(1944, Avery el al) The DNA of S-type bacteria can transform R-type bacteria into the same S-type

6 Discovery of pneumococcal transformation (Streptococcus pneumoniae), 1928 Frederick Griffith, 1928

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8 At this time a wholly unexpected result was obtained. Introduction of the transforming principles into the R cells results in acquisition by these bacteria of a new heritable characteristic- the ability to synthesize the capsular polysaccharide. Identify the transforming principle and the chemical nature of the genetic material would be known

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12 Avery MacLeod McCarty experiment confirmed Griffith finding, 1944 Oswald Avery Colin MacLeod Transforming principle DNA that is taken up by a bacterium and whose expression then changes the properties of the recipient cell. Maclyn McCarty

13 Avery Experiments

14 Alfred Day Hershey finally confirmed DNA is genetic material!!!! (1952) awarded the Nobel Prize in Physiology or Medicine in 1969 Phage infection showed that DNA is the genetic material of viruses. When the DNA and protein components of bacteriophages are labeled with different radioactive isotopes, only the DNA is transmitted to the progeny phages produced by infecting bacteria Awarded the Nobel Prize in Physiology or Medicine in 1969, shared with Luria and Delbrück for their discovery on the replication of viruses and their genetic structure.

15 J Gen Physiol September 20; 36(1):

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17 1.4 Polynucleotide Chains Have Nitrogenous Bases Linked to a Sugar Phosphate Backbone A nucleoside consists of a purine or pyrimidine base linked to the 1 carbon of a pentose sugar. The difference between DNA and RNA is in the group at the 2 position of the sugar. DNA has a deoxyribose sugar (2 H); RNA has a ribose sugar (2 OH).

18 Finding DNA structure In the late 1940, same era scientific community realized that DNA included different amounts of the four bases adenine(a), thymine(t), guanine(g), and cytosine(c)

19 Finding DNA structure; Erwin Chargoff (August 11, ) In the DNA of each species he studies, the number of adenines approximately equaled the number of thymine, and the number of guanines approximately equaled the number of cytosine. In human DNA, for example, the four bases are present in these percentages: A=30.9% and T=29.4%; G=19.9% and C=19.8%. The A=T and G=C equalities, later known as Chargaff's rules, helped Watson and Crick to discover the structure of DNA. (1952)

20 DNA mixture Measured by spectrometer Chromatography

21 Finding DNA structure In order to figure out (A,T,C,G) that the two strands run in opposite direction (25 July April 1958) Rosalind Franklin(1953) used X-ray diffraction to understand the physical structure of the DNA molecule Model building had been applied successfully in the elucidation of the structure of the alpha helix A rule only living persons can be nominated for the Nobel Prize

22 Maurice Wilkins and Rosalind Franklin DNA is a helix with two regular periodicities of 3.4A and 34A along the axis of the molecule

23 DNA is a helix with two regular periodicities of 3.4A and 34A along the axis of the molecule. How does this related to Chargaff d base ratios and to the actual structure of DNA?

24 Concluded the DNA-Helix, Nature, 4/25/1953, title Molecular structure of Nucleic Acid by Watson and Crick -The two strands of the double helix are anti-parallel, which means that they run in opposite directions. The base of the two polynucleotides interact by hydrogen bonding -The sugar-phosphate backbone is on the outside of the helix, and the bases are on the inside Nobel Prize in Physiology or Medicine in 1962.

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26 This structure has novel features which are of considerable biological interest!!!

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28 1. The double helix comprise two polynucleotides 2. The nitrogenous bases are stacked on the inside of the helix 3. The bases of the two polynucleotides interact by hydrogen bonding 4. Ten base pairs occur per turn of the helix 5. The two strands of the double helix are antiparallel

29 The nucleotides of DNA A nucleotide consists of a nucleoside linked to a phosphate group on either the 5 or 3 carbon of the (deoxy)ribose. Nucleoside Nucleoside Nucleotide H: deoxy- A C B D Nucleotide A: deoxythymidine-5 -phosphate B: deoxycytidine-5 -phosphate C: deoxyadenosine-5 -phosphate D: deoxyguanosine-5 -phosphate

30 Purine Pyrimidine

31 A hydrogen bond (very weak interaction) is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine

32 RNA: Template for protein DNA contains the four bases adenine, guanine, cytosine, and thymine; RNA has uracil instead of thymine. OH H Ribose Deoxyribose

33 1.4 Polynucleotide Chains Have Nitrogenous Bases Linked to a Sugar Phosphate Backbone Successive (deoxy)ribose residues of a polynucleotide chain are joined by a phosphate group between the 3 carbon of one sugar and the 5 carbon of the next sugar. One end of the chain (conventionally written on the left) has a free 5 end and the other end of the chain has a free 3 end.

34 1.6 DNA Is a Double Helix The B-form of DNA is a double helix consisting of two polynucleotide chains that run antiparallel. FIGURE 11: A polynucleotide chain

35 1.6 DNA Is a Double Helix The nitrogenous bases of each chain are flat purine or pyrimidine rings that face inward and pair with one another by hydrogen bonding to form only A-T or G-C pairs. complementary Base pairs that match up in the pairing reactions in double helical nucleic acids (A with T in DNA or with U in RNA, and C with G).

36 FIGURE 12: Flat base pairs lie perpendicular to the sugar-phosphate backbone

37 1.6 DNA Is a Double Helix The diameter of the double helix is 20 Å, and there is a complete turn every 34 Å, with ten base pairs per turn (~10.4 base pairs per turn in solution). The double helix has a major (wide) groove and a minor (narrow) groove.

38 DNA replication??

39 The Meselson - Stahl Experiment Nitrogen is a major constituent of DNA. (isotope N15 and N14) If conservative replication, equal amounts of DNA of the higher and lower densities would result in (but no DNA of an intermediate density) Wrong Semiconservative replication would result in double-stranded DNA with one strand of 15 N DNA, and one of 14 N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of 15 N and 14 N DNA----- Right!!!! If dispersive replication occurred, these would have resulted in a single density, lower than the intermediate density of the one-generation cells, but still higher than cells grown only in 14 N DNA medium, so dispersive replication was inconsistent --Wrong

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41 Meselson - Stahl

42 1.7 DNA Replication Is Semiconservative semiconservative replication DNA replication accomplished by separation of the strands of a parental duplex, each strand then acting as a template for synthesis of a complementary strand. The sequences of the daughter strands are determined by complementary base pairing with the separated parental strands. FIGURE 14: Base pairing provides the mechanism for replicating DNA

43 1.7 DNA Replication Is Semiconservative The Meselson Stahl experiment used heavy isotope labeling to show that the single polynucleotide strand is the unit of DNA that is conserved during replication. Replication of DNA is semiconservative

44 1.8 Polymerases Act on Separated DNA Strands at the Replication Fork Replication of DNA is undertaken by a complex of enzymes that separate the parental strands and synthesize the daughter strands. denaturation In DNA, this involves the separation of the two strands due to breaking of hydrogen bonds between bases. renaturation The reassociation of denatured complementary single strands of a DNA double helix.

45 1.8 Polymerases Act on Separated DNA Strands at the Replication Fork The replication fork is the point at which the parental strands are separated. The enzymes that synthesize DNA are called DNA polymerases. FIGURE 16: The replication fork

46 1.8 Polymerases Act on Separated DNA Strands at the Replication Fork Nucleases are enzymes that degrade nucleic acids; they include DNases and RNases and can be categorized as endonucleases or exonucleases. FIGURE 17: An endonuclease cleaves a bond within a nucleic acid FIGURE 18: An exonuclease removes bases one at a time

47 1.9 Genetic Information Can Be Provided by DNA or RNA Cellular genes are DNA, but viruses may have genomes of RNA. DNA is converted into RNA by transcription, and RNA may be converted into DNA by reverse transcription. RNA polymerase An enzyme that synthesizes RNA using a DNA template (formally described as DNAdependent RNA polymerases).

48 1.9 Genetic Information Can Be Provided by DNA or RNA central dogma Information cannot be transferred from protein to protein or protein to nucleic acid, but can be transferred between nucleic acids and from nucleic acid to protein. The translation of RNA into protein is unidirectional. FIGURE 19: The central dogma

49 1.10 Nucleic Acids Hybridize by Base Pairing Heating causes the two strands of a DNA duplex to separate. The melting temperature (T m ) is the midpoint of the temperature range for denaturation. Complementary single strands can renature or anneal when the temperature is reduced. FIGURE 23: Denatured single strands of DNA can renature to give the duplex form

50 1.10 Nucleic Acids Hybridize by Base Pairing Denaturation and renaturation/hybridization can occur with DNA DNA, DNA RNA, or RNA RNA combinations. Hybridization can be intermolecular or intramolecular. FIGURE 22: Base pairing

51 1.10 Nucleic Acids Hybridize by Base Pairing The ability of two singlestranded nucleic acids to hybridize is a measure of their complementarity. FIGURE 24: Filter hybridization

52 1.11 Mutations Change the Sequence of DNA All mutations are changes in the sequence of DNA. Mutations may occur spontaneously or may be induced by mutagens. FIGURE 25: Mutation rates

53 1.12 Mutations May Affect Single Base Pairs or Longer Sequences A point mutation changes a single base pair. Point mutations can be caused by the chemical conversion of one base into another or by errors that occur during replication. FIGURE 26: Mutations can be induced by chemical modification of a base

54 1.12 Mutations May Affect Single Base Pairs or Longer Sequences A transition replaces a G-C base pair with an A-T base pair or vice versa. A transversion replaces a purine with a pyrimidine, such as changing A-T to T-A. Insertions and/or deletions can result from the movement of transposable elements.

55 1.13 The Effects of Mutations Can Be Reversed Forward mutations alter the function of a gene, and back mutations (or revertants) reverse their effects. Insertions can revert by deletion of the inserted material, but deletions cannot revert. Point mutations and insertions can revert, but deletions cannot revert

56 1.13 The Effects of Mutations Can Be Reversed true reversion A mutation that restores the original sequence of the DNA. second-site reversion A second mutation suppressing the effect of a first mutation within the same gene. Suppression occurs when a mutation in a second gene bypasses the effect of mutation in the first gene.

57 1.14 Mutations Are Concentrated at Hotspots The frequency of mutation at any particular base pair is statistically equivalent, except for hotspots, where the frequency is increased by at least an order of magnitude. FIGURE 29: Spontaneous mutations are concentrated at a hotspot

58 1.15 Many Hotspots Result from Modified Bases A common cause of hotspots is the modified base 5- methylcytosine, which is spontaneously deaminated to thymine. A hotspot can result from the high frequency of change in copy number of a short, repeated sequence. FIGURE 30: Deamination

59 1.16 Some Hereditary Agents Are Extremely Small Some very small hereditary agents do not code for polypeptide, but consist of RNA or protein with heritable properties. viroid A small infectious nucleic acid that does not have a protein coat. FIGURE 32: PSTV RNA

60 1.16 Some Hereditary Agents Are Extremely Small prion A proteinaceous infectious agent that behaves as an inheritable trait even though it contains no nucleic acid. One example is PrPSc (Prions pecific epitope), the agent of scrapie in sheep and bovine spongiform encephalopathy.

61 Genes

62 Genes are units of biological information and inheritance Biological information 1) Construct a living, functioning example 2) Responsible for the visible characteristic (Eye color) 3) Biological activities Biological information is encoded in the nucleotide sequence of a DNA molecule, and that complementary base paring enables DNA molecules to be replicated: Gene Expression Gene Expression: The information contained in a gene is utilized by the cell 1) Nature of individual gene blue eye, Red 2) Small alternation in the nucleotide sequence inactivate a gene or alter the precise nature of the information 3) Biological activities

63 Genes contain instructions for making RNA and protein molecules - RNA Transcription - Protein synthesis is the key to expression of biological information 1) Proteins with different amino acid sequences can have quite different chemical properties that enable them to play a variety role 2) Construct structural protein such as collagen (Rigid Protein) 3) Flexible protein which enable to move around 4) Enzyme which consist of amino acid enabling them to catalyze the multitude of cellular reaction 5) Transport protein (Hemoglobin) 6) Regulatory protein that control cellular activity such as insulin

64 Genes and biological information

65 How a complete set of genes is passed to the daughters when the parent cell divide Study chromosome The inheritance of gene during sexual reproduction Evolution change over time How is a complete set of genes passed to the daughter cells when the parent cell divides?

66 Polynucleotides as their coding strand: a polynucletide that is the coding strand for one gene may be the noncoding strand for a second gene. 150 bp combination?? Coding sequence? Non Coding sequence? The biological information of a gene is in fact carried by just one of the two polynucleotides of the double helix

67 Gene Expression- Protein Synthesis Protein synthesis is the key to expression of biological information - Structural Protein: bones, cartilage - Contractile Proteins: myosin, actin - Enzymes - Transport Proteins - Regulatory Proteins: hormone, insulin - Protective Protein: Immunoglobulin - Storage Protein: egg