dna as the genetic material

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dna as the genetic material

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1. DNA as the Genetic Material Genetic information is defined as information contained in genes which, when passed to a new generation, influences the form and characteristics of the offspring Genetic material must be capable of replication, information storage, information expression and variation (mutation) Until 1944 it was not known which component of chromosomes was the genetic material Until 1953 it was not known how DNA could encode genetic information

2. Central Dogma of Molecular Genetics

3. Early Studies Beginning with the earliest observations concerning heredity, genetic material was assumed to exist Until the 1940s proteins were considered by geneticists to be the best candidates Very abundant in cells and did nifty things Nucleic acids were similar, boring and just a couple of nucleotides connected to each other…

4. Discovery of DNA 1868 by Friedrick Miescher, a Swiss chemist Called in nuclein since it was from the nucleus Had large amounts of phosphorous and no sulfur so was very different than protein

5. First Structure By 1910 actual components known (nucleotides) Phoebus Levene proposed a tetranucleotide structure for DNA

6. So… If DNA was a single covalently bonded tetranucleotide structure then it couldn’t easily encode information Proteins, on the other hand, had 20 different amino acids and could have lots of variation Most geneticists focused on “transmission genetics” and passively accepted proteins as being the likely genetic material

7. First Real Break 1927, Frederick Griffith Studied Pneumococcus (then became Diplococcus pneumoniae, then became Streptococcus pneumoniae) IIR strain was avirulent and lacked a lipopolysaccharide (LPS) capsule, growing in rough-shaped colonies on a plate IIIS strain was virulent, possessed a lipopolysaccharide capsule and could kill mice, and made round colonies

8. Frederick Griffith The Experiment Inject mouse with strain S ? mouse dies Inject mouse with strain R? mouse lives Inject with heat-killed strain S? mouse lives Inject with h-k S and live R ? mouse dies, and live S strain can be recovered from dead mouse Griffith concluded that the live R had been transformed to S by picking up the genetic material encoding the LPS from the dead S and using that material to repair the damaged/lost gene in the R strain

10. Griffith’s Experiment Called the material in the dead S cells that allowed for the R?S transformation the transforming principle First assay for the genetic material

11. Avery, McCarty and MacLeod After 10 yrs of effort published work using Griffith’s approach to assay for the genetic material Used Cell-free extract of S cells From 75 liters of cell culture obtained 10-25 mg of “active factor Proteases, RNases, DNases, etc. “The evidence presented supports the belief that a nucleic acid of the desoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type III”

12. Avery, McCarty and MacLeod

13. Harriet Taylor 1949 follow-up Studied strain R and strain ER (extremely rough) Showed DNA from R could convert ER strains to R strains and then DNA from S strains could convert R to S strains Conclusion: R strains could be both donor and recipients in transformation experiments

14. Hershey Chase Experiment Alfred Hershey and Martha Chase, 1952 Evidence that DNA is the genetic material Simple model system using T2 bacteriophage and radioactive materials

15. Life Cycle of T-Even Phage Phage made of DNA and protein What enters cell and allows production of new phage?

16. Hershey Chase Experiment T2 Phage, E. coli, and 35S, Waring blender 32P04 goes into DNA 35S04 goes into proteins Experiment Grow phage on cells cultured in 32P04 and 35S04 Infect new cells (not radioactive) with radioactive phage After various times place in Waring blender, centrifuge and measure radioactivity in cells plus plate them out to determine whether successfully infected by phage Allow some to complete life cycle and measure radioactivity levels of progeny phage

17. Hershey-Chase Experiment Time course also reveals that entry of 32P into cells correlates with successful infection

18. Indirect Evidence for Eukaryotes DNA found “only” in nucleus, proteins all over cell DNA in chromosomes Ploidy correlated with DNA content

20. More Indirect Evidence: Mutagenesis Action spectrum of UV light for mutagenesis correlates well with the absorption spectrum of DNA UV light of 260 nm most mutagenic DNA absorption maximum is 260 nm Protein absorption maximum is 280 nm

21. Action and Absorption Spectra

22. RNA as Genetic Material Fraenkel-Conrat and Singer, 1956 Tobacco Mosaic Virus (TMV) and Holmes Ribgrass Virus (HRV) Closely related plant viruses made of an RNA molecule encased in a spiral of protein One coat protein could encapsulate the other RNA and still function properly during infection

23. RNA as Genetic Material

24. RNA Can Replicate Pace and Spiegelman, 1965, 1966 Phage Qb Isolated an RNA replicase enzyme that could replicate the Qb chromosome in vitro No DNA involved

25. Reverse Transcription Retroviruses (e.g. HIV, RSV) RNA chromosomes Convert to DNA by reverse transcriptase Insert DNA into host chromosome Transcribe new RNA copies

26. Nucleic Acid Structure DNA is a nucleic acid composed of nucleotides Nucleotides have a nitrogenous base, a pentose sugar and a phosphate group Bases are either pyrimidines (cytosine and thymine in DNA or C and uracil in RNA) or purines (adenine and guanine) Pentose sugar is either deoxyribose (DNA) or ribose (RNA) A base plus a sugar is a nucleoside, add phosphate for a nucleotide (nucleotides named by nucleoside plus number of phosphates – adenosine diphosphate) Sugar on C-1’ position, phosphate commonly on C-5’

27. Components of Nucleic Acids Purines Pyrimidines 5-carbon sugar phosphate

28. Nucleosides and Nucleotides

29. Nucleoside Diphosphates and Triphosphates

30. Polynucleotides Nucleotides of a single strand connected by covalent 5’-3’ phosphodiester bond Following Levene’s tetranucleotide hypothesis it was clearly shown that bases were not present in equimolar quantities and that DNA molecules were in fact quite large

31. Phosphodiester Bonds Phosphate is from phosphoric acid Hydroxyl groups on sugars represent alcohol Acid plus alcohol given ester Phosphate reacts with two –OH groups

32. Structure of DNA Structure of DNA should reveal how it works as the genetic material Intense study from 1940-1953 Chargaff, Wilkins, Franklin, Pauling, Watson, Crick and more… First to elucidate the correct structure gets the big one

33. Erwin Chargaff 1949-1953 Digested many DNAs and subjected products to chromatographic separation Results A = T, C = G A + G = C + T (purine = pyrimidine) A + T does not equal C + G Members of a species similar but different species vary in AT/CG ratio

35. Franklin and Wilkins X-ray diffraction analysis of DNA crystals Originally by William Astbury (1938)who detected a periodicity of 3.4 angstroms (1947) Pauling used data to propose a triple helix 1950-1953 Franklin (in Wilkins’ lab) confirmed 3.4 periodicity and noted uniform diameter of 20 angstroms (2 nm) Proposed no definitive model

36. X-ray Crystallography of DNA Franklin and Wilkins

37. Watson and Crick 1953 propose double helix model Right-handed double helix Chains antiparallel Bases lie flat, perpendicular to long axis of chain Bases pair by hydrogen bonds, A with T and C with G Two strands are complementary 10 bases per turn (34 angstroms) Now known to be 10.4 or 34.6 degrees turn per bp) Has a major and minor groove Is 20 angstroms in diameter

38. DNA Double Helix

39. Right vs. Left Handed Helices

40. Base Pairing Hydrogen bonds reversible Individually weak electrostatic bonds but collectively can be strong

41. Impact Article in Nature “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copy mechanism for the genetic material” Second paper 2 months later describes semiconservative replication and that mutations must change bases in DNA (information encoded in the bases and their order) DNA became the genetic material…

42. Alternative Forms of DNA DNA can exist in several conformational isomers B form is the “normal” conformation A form is found in high salt Probably not biologically relevant D and E forms (8 and 7 bp/turn respectively) DNA segments lacking guanine Z form Left-handed helix and 12 bp/turn (Z for zigzag) C-G base pairs only P form Phosphates to inside and bases more to outside Are P and/or Z biologically relevant???

43. Conformational Forms of DNA

44. Structure of RNA Ribose for deoxyribose, uracil for thymine RNA tends to be single stranded Can fold back to have secondary structure Can be double stranded in some phage/viruses Major classes of RNA Ribosomal RNA tRNA mRNA But there are several others…

45. Major Classes of RNA…but there are more S is for the Svedberg sedimentation coefficient

46. Other RNAs To be discussed in later chapters snRNAs Telomerase RNA siRNAs Antisense RNAs

47. Nucleic Acid Characterization Absorption Spectra Absorb light in ultraviolet range, most strongly in the 254-260 nm range Due to the purine and pyrimidine bases Useful for localization, characterization and quantification of samples

48. Nucleic Acid Characterization Sedimentation and density Can be characterized by sedimentation velocity (Svedberg coefficient, S) Sedimentation velocity centrifugation Related to MW and shape Or by buoyant density CsCl (DNA) or CsSO4 for RNA Sedimentation equilibrium centrifugation

49. Buoyant Density Centrifugation

50. Base Composition vs. Density G-C base pairs are more dense than A-T pairs

51. Denaturation of Nucleic Acids Denaturation involves the breaking of hydrogen bonds Disrupts the base stacking in the helix and lead to increased absorbance at 260 nm Hyperchomic shift By increasing temperature slowly and measuring absorbance at 260 nm as melting profile can be generated Temperature for midpoint of denaturation is called the Tm

52. Thermal Denaturation Increased G+C gives increased Tm 3 vs. 2 hydrogen bonds Increased ionic strength also increases Tm

53. Hybridization After nucleic acids are denatured they can be allowed to reform base pairs with complementary molecules Molecular hybridization Close but not perfect match required stringency Can involve DNA:DNA or DNA:RNA FISH, Southern transfer (blotting) and DNA microarray analyses involve hybridization

54. Hybridization

55. Fluorescent in situ Hybridization FISH Use DNA or RNA probes for hybridization Originally radioactive Now biotin and fluorescent dyes Cells/chromosomes fixed to slide before hybridization Can detect single copy genes

56. Reassociation Kinetics Denatured DNA duplexes can reassociate with complementary strands to reform duplex Chemical reaction, rate depends upon conditions including substrate concentration

57. Reassociation Kinetics

58. Reassociation Kinetics DNA concentration is routinely measured in micrograms per ml (mass/volume) But here the relevant concentration is copies of complementary DNA (not mass) per unit volume And this depends upon both the mass per volume and the size of the genome being studied

59. Reassociation Curves of Different DNAs

60. Genome Size vs. C0t1/2

61. C0t Analyses Previous curves were for genomes generally lacking repetitive sequence regions Al or nearly all sequences present at one copy per genome What happens to the C0t analyses when genomes have repetitive sequences? Single copy, middle and highly repetitive

62. C0t Analyses

63. Gel Electrophoresis Agarose or polyacrylamide gels DNA is negatively charged and migrates toward positive pole when placed in an electric field Smaller fragments move through the gel matrix more quickly and therefore migrate faster per unit of time Extremely common method for characterizing and purifying DNA fragments Including DNA sequencing procedures

64. Gel Electrophoresis

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