1. The Molecular Basis of Inheritance13
The Molecular Basis of Inheritance

2. Miescher Discovered DNA
Johann Miescher investigated the nucleus
Isolated DNA

3. The Search for the Genetic Material: Scientific Inquiry
T. H. Morgan’s showed that genes are located on chromosomes,
DNA and protein became candidates for the genetic material 3

4. Griffith Discovers Transformation
1928
Attempting to develop a vaccine
2 strains of Streptococcus pneumoniae
Rough strain - harmless
Smooth strain - pathogenic

5. Mice injected with live cells of harmless strain R.
Mice live. No live R cells in their blood.
Mice injected with live cells of killer strain S.
Mice die. Live S cells in their blood.
Mice injected with heat-killed S cells.
Mice live. No live S cells in their blood.
Mice injected with live R cells plus heat-killed S cells.
Mice die. Live S cells in their blood.
Stepped Art
Fig. 13-3, p.208

6. Experiment Mixture of heat-killed S cells and living R cells Living
Figure 13.2
Experiment
Mixture of
heat-killed
S cells and
living R cells
Living
S cells
(control)
Living
R cells
(control)
Heat-killed
S cells
(control)
Figure 13.2 Inquiry: Can a genetic trait be transferred between different bacterial strains?
Results
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells 6

7. Oswald Avery What is the transforming material?
Cell with all proteins digested transformed bacteria
Cell with DNA digested lost transforming ability

8. Evidence That Viral DNA Can Program Cells
More evidence for DNA as the genetic material came from studies of bacteriophages
Animation: Phage T2 Reproduction 8

9. Bacteriophages Viruses that infect bacteria Consist of protein and DNA
bacterial cell wall
plasma membrane
cytoplasm

10. Phage head Tail sheath Tail fiber DNA 100 nm Bacterial cell
Figure 13.3
Phage
head
Tail
sheath
Tail fiber
DNA
Figure 13.3 Viruses infecting a bacterial cell
100 nm
Bacterial
cell 10

11. Hershey & Chase’s Experiments
1952 Allowed radioactively labeled viruses to infect bacteria

12. Hershey and Chase Results
35S remains
outside cells
virus particle
labeled with 35S
DNA (blue)
being injected into bacterium
virus particle
labeled with 32P
35P remains
inside cells
DNA (blue)
being injected into bacterium
Fig. 13-4ab, p.209

13. 1950, Chargaff’s rules DNA base composition varies between species
In any species the number of A and T bases is equal and the number of G and C bases is equal 13

14. Building a Structural Model of DNA: Scientific Inquiry
Maurice Wilkins and Rosalind Franklin studied molecular structure using X-ray crystallography
Franklin produced image of DNA using this technique 14

15. (b) Franklin’s X-ray diffraction photograph of DNA
Figure 13.6
Figure 13.6 Rosalind Franklin and her X-ray diffraction photo of DNA
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
photograph of DNA 15

16. Building a Structural Model of DNA: Scientific Inquiry
James Watson and Francis Crick were first to determine the structure of DNA 16

17. Figure 13.1
Figure 13.1 How was the structure of DNA determined? 17

18. Sugar– Nitrogenous bases phosphate backbone 5 end Thymine (T)
Figure 13.5
Sugar–
phosphate
backbone
Nitrogenous bases
5 end
Thymine (T)
Adenine (A)
Cytosine (C)
Figure 13.5 The structure of a DNA strand
Guanine (G)
3 end
DNA
nucleotide 18

19. Sugar (deoxyribose) Nitrogenous base
Figure 13.5a
Phosphate
3 end
Sugar
(deoxyribose)
Figure 13.5a The structure of a DNA strand (part 1: nucleotide)
DNA
nucleotide
Nitrogenous
base 19

20. (b) Partial chemical structure (c) Space-filling model
Figure 13.7
5 end C G C G
Hydrogen bond
3 end G C G C T A
3.4 nm T A G C G C C G A T
1 nm C G T A C G G C C G A T
Figure 13.7 The double helix A T
3 end A T
0.34 nm T A
5 end
(a) Key features of
DNA structure
(b) Partial chemical structure
(c) Space-filling
model 20

21. Structure of Nucleotides in DNA
Know it

22. Purine  purine: too wide
Figure 13.UN02
Purine  purine: too wide
Pyrimidine  pyrimidine: too narrow
Figure 13.UN02 In-text figure, purines and pyrimidines, p. 250
Purine  pyrimidine: width
consistent with X-ray data 22

23. Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G)
Figure 13.8
Sugar
Sugar
Adenine (A)
Thymine (T)
Figure 13.8 Base pairing in DNA
Sugar
Sugar
Guanine (G)
Cytosine (C) 23

24. Nucleotides & stuff nitrogen-containing base
A nucleoside is one nitrogenous base plus one sugar
A nucleotide is one nucleoside plus one or more phosphate groups
Fig. 13-5, p.210

25. Nucleotide Bases Purines Pyrimidines thymine (T) base with a adenine
single-ring
structure
adenine A
base with a
double-ring
structure
Purines
Pyrimidines
guanine
(G)
base with a
double-ring
structure
cytosine
(C)
base with a
single-ring
structure
Fig. 13-5, p.210

26. Nucleotide Bases thymine (T) base with a adenine single-ring A
structure
adenine A
base with a
double-ring
structure
guanine
(G)
base with a
double-ring
structure
cytosine
(C)
base with a
single-ring
structure
Fig. 13-5, p.210

27. Nucleotides have a 3’ end with
an -OH group and a 5’ end with a phosphate group (3 P’s on a free nucleotide) 5’ 3’
Fig. 13-5, p.210

28. DNA Replication: Getting Started
Replication begins at sites called origins of replication, opening up a replication “bubble”
each end of a bubble is a replication fork where the DNA is unwound by helicases
Animation: DNA Replication Overview
Animation: Origins of Replication 28

29. Single-strand binding proteins
Figure 13.12
Primase
Topoisomerase 3
RNA
primer 5 3 5
Replication
fork 3
Helicase
Figure Some of the proteins involved in the initiation of DNA replication 5
Single-strand binding
proteins 29

30. Origin of replication Replication fork Replication fork
Figure 13.13
(a) Origin of replication in an E. coli cell
(b) Origins of replication in a eukaryotic
cell
Parental
(template) strand
Origin of
replication
Origin of
replication
Double-stranded
DNA molecule
Daughter
(new) strand
Daughter (new)
strand
Parental (template)
strand
Replication
fork
Double-
stranded
DNA
molecule
Replication
bubble
Bubble
Replication fork
Two
daughter DNA
molecules
Two daughter DNA molecules
Figure Origins of replication in E. coli and eukaryotes
0.25 m
0.5 m 30

31. Figure 13.13aa
0.5 m
Figure 13.13aa Origins of replication in E. coli and eukaryotes (part 1a: E. coli, TEM) 31

32. Multiple replication bubbles form and fuse, speeding up copying32

33. Origin of replication Replication fork
Figure 13.13b
(b) Origins of replication in a eukaryotic cell
Origin of
replication
Double-stranded
DNA molecule
Daughter (new)
strand
Parental (template)
strand
Replication fork
Bubble
Figure 13.13b Origins of replication in E. coli and eukaryotes (part 2: eukaryotes)
0.25 m
Two daughter DNA molecules 33

34. Synthesizing a New DNA Strand
DNA polymerases only add nucleotides to existing chain - cannot initiate synthesis
initial nucleotide strand is a short RNA primer
new DNA strand will start from the 3 end of the RNA primer
Then DNA polymerases add nucleotides 34

35. DNA poly- merase Pyro- phosphate
Figure 13.14
New strand
Template strand 5 3 5 3
Sugar A T A T
Base
Phosphate C G C G
DNA
poly-
merase G C G C 3 A T A T
Figure Addition of a nucleotide to a DNA strand P P P P i P C 3 C
Pyro-
phosphate
Nucleotide 5 5 2 P i 35

36. A Closer Look at Strand Assembly
Energy for strand assembly provided by removal of two phosphate groups from free nucleotides
newly
forming
DNA
strand
one parent
DNA strand

37. Antiparallel Elongation
Strands can only be assembled in the 5’ to 3’ direction
Reiji Okazaki discovered that strand assembly is continuous on just one parent strand

38. gaps are joined by DNA ligase
After formation of Okazaki fragments, DNA polymerase I removes RNA primers and replaces nucleotides with DNA
gaps are joined by DNA ligase
Animation: Lagging Strand
Animation: DNA Replication Review 38

39. Overall directions of replication
Figure 13.15a
Overview
Leading
strand
Origin of replication
Lagging
strand
Primer
Leading
strand
Lagging strand
Overall
directions
of replication
Figure 13.15a Synthesis of the leading strand during DNA replication (part 1) 39

40. Overall directions of replication
Figure 13.16a
Overview
Lagging
strand
Origin of replication
Leading
strand
Lagging
strand
Leading
strand
Overall directions
of replication
Figure 13.16a Synthesis of the lagging strand (part 1) 40

41. Primase makes RNA primer. Template strand 1 3 5 3 5
Figure 13.16b-1 1
Primase makes
RNA primer. 3 5 3
Template
strand 5
Figure 13.16b-1 Synthesis of the lagging strand (part 2, step 1) 41

42. Primase makes RNA primer. Template strand RNA primer DNA pol III
Figure 13.16b-2 1
Primase makes
RNA primer. 3 5 3
Template
strand 5
RNA primer
for fragment 1 2
DNA pol III
makes Okazaki
fragment 1. 3 5 3 5
Figure 13.16b-2 Synthesis of the lagging strand (part 2, step 2) 42

43. Primase makes RNA primer. Template strand RNA primer DNA pol III
Figure 13.16b-3 1
Primase makes
RNA primer. 3 5 3
Template
strand 5
RNA primer
for fragment 1 2
DNA pol III
makes Okazaki
fragment 1. 3 5 3 5
Figure 13.16b-3 Synthesis of the lagging strand (part 2, step 3) 3
DNA pol III
detaches. 3
Okazaki
fragment 1 5 3 5 43

44. RNA primer for fragment 2
Figure 13.16c-1
RNA primer for fragment 2
Okazaki
fragment 2 5 4
DNA pol III
makes Okazaki
fragment 2. 3 3 5
Figure 13.16c-1 Synthesis of the lagging strand (part 3, step 1) 44

45. RNA primer for fragment 2
Figure 13.16c-2
RNA primer for fragment 2
Okazaki
fragment 2 5 4
DNA pol III
makes Okazaki
fragment 2. 3 3 5 5 5
DNA pol I
replaces RNA
with DNA. 3 3 5
Figure 13.16c-2 Synthesis of the lagging strand (part 3, step 2) 45

46. RNA primer for fragment 2
Figure 13.16c-3
RNA primer for fragment 2
Okazaki
fragment 2 5 4
DNA pol III
makes Okazaki
fragment 2. 3 3 5 5 5
DNA pol I
replaces RNA
with DNA. 3 3 5 6
DNA ligase forms
bonds between
DNA fragments.
Figure 13.16c-3 Synthesis of the lagging strand (part 3, step 3) 5 3 3 5
Overall direction of replication 46

47. Leading strand template Single-strand binding proteins Leading strand
Figure 13.17
Overview
Origin of replication
Leading strand
template
Leading strand
Lagging
strand
Single-strand
binding proteins
Lagging strand
Leading strand
Overall directions
of replication
Leading strand
Helicase
DNA pol III 5 3
Primer 5 3
Primase 3
Parental DNA
Lagging strand
DNA pol III
Figure A summary of bacterial DNA replication 5
Lagging strand
template 3
DNA pol I
DNA ligase 5 3 5 47

48. DNA pol III Parental DNA Leading strand 5 5 3 3 3 5 3 5
Figure 13.18
DNA pol III
Parental DNA
Leading strand 5 5 3 3 3 5 3 5
Connecting
proteins
Helicase
Lagging
strand
template 3 5
Figure A current model of the DNA replication complex
DNA
pol III
Lagging strand 3 5 48

49. 5 3 3 5 Nuclease 5 3 3 5 DNA polymerase 5 3 3 5 DNA ligase
Figure 5 3 3 5
Nuclease 5 3 3 5
DNA
polymerase 5 3
Figure Nucleotide excision repair of DNA damage (step 3) 3 5
DNA ligase 5 3 3 5 49

50. Discovery of Restriction Enzymes
Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack
Discovered bacteria have an enzyme that chops up viral DNA

51. Specificity of Cuts
Restriction enzymes cut DNA at a specific base sequence
Cut leaves “sticky ends”
Number of cuts made in DNA will depend on number of times the “target” sequence occurs

52. Restriction enzyme cuts the sugar-phosphate backbones.
Figure
Restriction site 5 3
DNA G A A T T C C T T A A G 3 5 1
Restriction enzyme cuts
the sugar-phosphate
backbones. 3 5 5 3 G A A T T C C T T A A G 5 3 3 5
Sticky end
Figure Using a restriction enzyme and DNA ligase to make recombinant DNA (step 1) 52

53. Restriction enzyme cuts the sugar-phosphate backbones.
Figure
Restriction site 5 3
DNA G A A T T C C T T A A G 3 5 1
Restriction enzyme cuts
the sugar-phosphate
backbones. 3 5 5 5 A 3 G A T T C T T A A C G 5 3 3 5
Sticky end 5 2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs. A A T 3 T C G 3 5
Figure Using a restriction enzyme and DNA ligase to make recombinant DNA (step 2) 5 3 5 3 5 3 G A A T T C G A A T T C C T T A A G C T T A A G 3 5 3 5 3 5
One possible combination 53

54. Restriction enzyme cuts the sugar-phosphate backbones.
Figure
Restriction site 5 3
DNA G A A T T C C T T A A G 3 5 1
Restriction enzyme cuts
the sugar-phosphate
backbones. 3 5 5 3 G A A T T C C T T A A G 5 3 3 5
Sticky end 5 2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs. A A T 3 T C G 3 5
Figure Using a restriction enzyme and DNA ligase to make recombinant DNA (step 3) 5 3 5 3 5 3 G A A T T C G A A T T C C T T A A G C T T A A G 3 5 3 5 3 5 3
DNA ligase
seals the strands.
One possible combination 5 3 3
Recombinant DNA molecule 5 54

55. Gel Electrophoresis DNA placed at one end of gel
current is applied to gel
DNA has negative charge … moves toward + end of gel
Smaller molecules move faster than larger ones

56. (a) Negatively charged DNA molecules will move
Figure 13.24a
Power
source
Mixture of
DNA mol-
ecules of
different
sizes
Cathode
Anode
Wells
Gel
Figure 13.24a Gel electrophoresis (part 1: art)
(a) Negatively charged DNA molecules will move
toward the positive electrode. 56

57. Restriction fragments
Figure 13.24b
Figure 13.24b Gel electrophoresis (part 2: photo)
Restriction fragments
(b) Shorter molecules are impeded less than
longer ones, so they move faster through the gel. 57

58. Gel Electrophoresis
Fig. 16-9b, p.249

59. Amplifying DNA 2 Methods insert into fast-growing microorganisms
Polymerase chain reaction (PCR)

60. Polymerase Chain Reaction
Double-stranded
DNA to copy
DNA heated to
90°– 94°C
Primers added to
base-pair with
ends
Mixture cooled;
base-pairing of
primers and ends
of DNA strands
DNA polymerases
assemble new
DNA strands
Stepped Art
Fig. 16-6, p. 256

61. Polymerase Chain Reaction
Sequence to be copied is heated
Primers added - bind to ends of single strands
DNA polymerase uses free nucleotides to create complementary strands
Doubles number of copies

62. DNA Sequencing… Reaction Mixture
Copies of DNA to be sequenced
Primer
DNA polymerase
Standard nucleotides
Modified nucleotides

63. Nucleotides for Sequencing
Standard nucleotides (A, T, C, G)
Modified versions
Labeled to fluoresce (different color for each base)
Structure causes DNA to stop synthesis when added

64. Reactions Proceed
Nucleotides added to create complementary strands - each ends at tagged nucleotide
Result - millions of copies of varying length with 1 flourescent base at the end

65. Recording the Sequence
T C C A T G G A C C
T C C A T G G A C
Recording the Sequence
T C C A T G G A
T C C A T G G
T C C A T G
T C C A T
T C C A
electrophoresis
gel
T C C
DNA placed on gel
Fragments move off gel in size order; pass through UV laser beam
Color each fragment fluoresces is recorded
T C
one of the many fragments of
DNA migrating
through the gel T
one of the DNA fragments
passing through a laser beam
after moving through the gel
T C C A T G G A C C A

66. Recording the Sequence
Fig. 16-8b, p.248

67. RFLP DNA Fingerprinting background information Tandem Repeats
Short DNA regions that differ substantially among people
Many sites in genome where tandem repeats occur

68. RFLPs Restriction fragment length polymorphisms
DNA from areas with tandem repeats is cut with restriction enzymes
Variation in the amount of repeat creates variation in fragment size
Variation is detected by gel electrophoresis

69. Using Plasmids Plasmid is small circle of bacterial DNA
Foreign DNA can be inserted into plasmid
Forms recombinant plasmids
Plasmid is a cloning vector
Can deliver DNA into another cell

70. Plasmids
Fig. 16-3a, p.244

71. Plasmids
Fig. 16-3b, p.244

72. Host cell grown in culture to form a clone of
Figure 13.22
Bacterium 1
Gene inserted
into plasmid
Cell containing gene
of interest
Bacterial
chromosome
Plasmid
DNA of chromosome
(“foreign” DNA)
Recombinant
DNA (plasmid)
Gene of interest 2
Plasmid put into
bacterial cell
Recombinant
bacterium 3
Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest
Protein expressed
from gene of interest
Copies of gene
Protein harvested
Figure An overview of gene cloning and some uses of cloned genes 4
Basic
research
and various
applications
Gene for pest resistance
inserted into plants
Human growth hormone
treats stunted growth
Gene used to alter bacteria
for cleaning up toxic waste
Protein dissolves blood clots
in heart attack therapy 72

73. Cell containing Bacterium gene of interest Gene inserted into plasmid
Figure 13.22a
Cell containing
gene of interest
Bacterium 1
Gene inserted
into plasmid
Bacterial
chromosome
Plasmid
DNA of
chromosome
(“foreign” DNA)
Recombinant
DNA (plasmid)
Gene of interest 2
Plasmid put into
bacterial cell
Recombinant
bacterium
Figure 13.22a An overview of gene cloning and some uses of cloned genes (part 1: steps) 3
Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Gene of
interest
Protein expressed
from gene of interest 73

74. Gene for pest resistance inserted into plants Human growth hormone
Figure 13.22b
Gene of
interest
Protein expressed
from gene of interest
Copies of gene
Protein harvested 4
Basic
research
and various
applications
Gene for pest resistance
inserted into plants
Human growth hormone
treats stunted growth
Figure 13.22b An overview of gene cloning and some uses of cloned genes (part 2: applications)
Gene used to alter bacteria
for cleaning up toxic waste
Protein dissolves blood clots
in heart attack therapy 74