DNA as the Genetic Material

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

• Tetranucleotide repeat of ATCG
• Own data showed nucleotides not in 1111 ratio
• Differences probably experimental error

6
So

• If DNA was a single covalently bonded
  tetranucleotide structure then it couldnt 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

9
(No Transcript)
10
Griffiths 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
  Griffiths 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

19
(No Transcript)
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 Levenes 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

34
(No Transcript)
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 RNAbut 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 GC 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 DNADNA or DNARNA
• 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