Gen Bio II BIO102

1. Chapter 16
BIOL 102
The
Molecular
Basis of
Inheritance
Rob Swatski
Assoc. Prof. Biology
HACC – York Campus

2. Overview: Life’s Operating Instructions
1953: James Watson & Francis Crick
- double-helix model
- structure of deoxyribonucleic acid (DNA)
DNA directs development of traits:
- biochemical
- anatomical
- physiological
- behavioral

4. The Search for the Genetic Material
After Morgan’s research on genes & chromosomes,
DNA & protein became likely candidates for
the genetic material
The key factor was choosing appropriate
experimental organisms
The role of DNA in heredity was first discovered
by studying bacteria and the viruses that infect
them

5. 1928: Frederick Griffith worked with 2 bacterial
strains:
one pathogenic & one harmless
When he mixed heat-killed remains of the pathogenic
strain with living cells of the harmless strain:
some living cells became pathogenic
He referred to this as transformation
- we now define it as a change in genotype &
phenotype due to assimilation of foreign DNA

6. Living S cells
(control)
Living R cells
(control)
Heat-killed S cells
(control)
Mixture of heat-
killed S cells &
living R cells
Mouse diesMouse dies Mouse healthy Mouse healthy
Living S cells
RESULTS
EXPERIMENT

7. 1944: Avery, McCarty, & MacLeod announced
that DNA was the transforming substance
- based on experimental evidence showing only
DNA helped transform harmless bacteria into
pathogens
- many biologists remained skeptical, mainly
because little was known about DNA

8. Bacterial
cell
Phage
capsid
Tail
sheath
Tail fiber
DNA
100nm
More Evidence: Bacteriophages (Phages)

9. 1952: Alfred Hershey & Barbara Chase
experiments:
- showed that DNA is the genetic material of T2 phage
- Results: only 1 of the 2 components of T2 (DNA or
protein) enters an E. coli cell during infection
Concluded that the phage’s injected DNA provides
the genetic information

10. EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1:
radioactive
sulfur (35S)
Batch 2:
radioactive
phosphorus
(32P)

11. EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive
DNA
Batch 1:
radioactive
sulfur (35S)
Batch 2:
radioactive
phosphorus
(32P)
Empty
protein
shell
Phage
DNA

12. EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive
protein
Radioactive DNA
Batch 1:
radioactive
sulfur (35S)
Batch 2:
radioactive
phosphorus
(32P)
Empty
protein
shell
Phage
DNA
Centrifuge
Centrifuge
Pellet
Pellet (bacterial
cells and contents)
Radioactivity
(phage
protein)
in liquid
Radioactivity
(phage DNA)
in pellet

13. Additional Evidence
It was known that DNA is a polymer of
nucleotides:
- nitrogenous base, a sugar, & a phosphate
group
1950: Erwin Chargaff showed DNA composition
varies between species
- this evidence of diversity made DNA a more
credible candidate for the genetic material

14. Sugar–phosphate
backbone
5 end
Nitrogenous
bases
Thymine (T)
Adenine (A)
Cytosine (C)
Guanine (G)
DNA nucleotide
Sugar (deoxyribose)
3 end
Phosphate

15. Chargaff’s Rules
In any species there is an equal number of:
A and T
&
G and C

16. Building a Structural Model of DNA
The next challenge was to relate DNA structure
with function
Maurice Wilkins & Rosalind Franklin: used X-ray
crystallography to study molecular structure
- took pictures of DNA

17. Rosalind Franklin Franklin’s X-ray diffraction
photograph of DNA

18. Franklin’s images of DNA enabled Watson to
deduce:
- shape: double helix
- width (double-stranded)
- spacing of N-bases

19. (c) Space-filling
model
Hydrogen bond 3 end
5 end
3.4 nm
0.34 nm
3 end
5 end
(b) Partial chemical
structure
(a) Key features of DNA
structure
1 nm

20. Watson & Crick built double helix models to match
the x-ray & chemical evidence
Franklin’s DNA structure hypothesis:
- 2 sugar-phosphate backbones
- paired nitrogenous bases in-between
Watson built a model in which the backbones
were antiparallel (their subunits run in opposite
directions)

21. Watson & Crick first thought bases paired “like
with like” (A-A, etc.)
- but this does not result in a uniform width
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow

22. Watson & Crick concluded that:
- Adenine (A) paired only with Thymine (T)
- Guanine (G) paired only with Cytosine (C)
This model explains Chargaff’s rules:
“in any organism the amount of
A = T and the amount of G = C”

23. Cytosine (C)
Adenine (A) Thymine (T)
Guanine (G)

24. DNA Replication and Repair
Watson & Crick noted that the specific base-pairing
suggested a possible DNA copying mechanism

25. The Basic Principle: Base Pairing to a
Template Strand
• Since the 2 strands of DNA are complementary,
each strand acts as a template for building a
new strand in replication
• In DNA replication, the parent molecule
unwinds & 2 new daughter strands are built
based on base-pairing rules

26. A T
GC
T A
TA
G C
(a) Parent molecule
A T
GC
T A
TA
G C
(c) “Daughter” DNA molecules, each
consisting of one parental strand
& one new strand
(b) Separation of
strands
A T
GC
T A
TA
G C
A T
GC
T A
TA
G C

27. Semiconservative Model of Replication
• Predicts that when a double helix replicates,
each daughter molecule will have one old strand
(derived or “conserved” from the parent
molecule) & one newly made strand
Competing models:
- Conservative model: the 2 parent strands
rejoin
- Dispersive model: each strand is a mix of old &
new

28. Parent cell
First
replication
Second
replication
(a) Conservative
model
(b) Semiconservative
model
(c) Dispersive model

29. • Experiments by Matthew Meselson & Franklin Stahl
supported the semiconservative model
• They labeled the nucleotides of the old strands with
a heavy isotope of N, while any new nucleotides
were labeled with a lighter isotope

30. EXPERIMENT
RESULTS
1
3
2
4
Bacteria
cultured in
medium
containing 15N
Bacteria
transferred to
medium
containing 14N
DNA sample
centrifuged after
20 min (after 1st
application)
DNA sample
centrifuged after
20 min (after 2nd
replication)
Less
dense
More
dense

31. • The 1st replication produced a band of hybrid DNA,
eliminating the conservative model
• A 2nd replication produced both light & hybrid DNA,
eliminating the dispersive model & supporting the
semiconservative model

32. CONCLUSION
First replication Second replication
Conservative model
Semiconservative
model
Dispersive model

33. • DNA replication is remarkable in its speed &
accuracy
• More than a dozen enzymes & other proteins
participate in DNA replication

34. Getting Started
• Replication begins at special sites called origins of
replication, where the 2 DNA strands are
separated, opening up a replication “bubble”
• A eukaryotic chromosome may have 100’s or
1000’s of origins of replication
• Replication proceeds in both directions from each
origin, until the entire molecule is copied

35. Origin of
replication Parental (template) strand
Daughter (new) strand
Replication fork
Replication
bubble
Double-stranded
DNA molecule
Two
daughter
DNA
molecules
(a) Origins of replication in
prokaryotes
0.5 µm

36. 0.25 µm
Origin of replication Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
Bubble Replication fork
Two daughter DNA molecules
(b) Origins of replication in
eukaryotes

37. • At the end of each replication bubble is a
replication fork, a Y-shaped region where new DNA
strands are elongating
• Helicases are enzymes that untwist the double helix
at the replication forks
• Single-strand binding proteins bind to & stabilize
single-stranded DNA until it can be used as a
template
• Topoisomerase corrects “overwinding” ahead of
replication forks by breaking, swiveling, & rejoining
DNA strands

38. Topoisomerase
Primase
RNA
primer
Helicase
Single-strand binding
proteins
5
3
5
53
3

39. • DNA polymerases cannot initiate synthesis of a
polynucleotide
- they can only add nucleotides to the 3 end
• The initial nucleotide strand is a short RNA primer
(5–10 nucleotides long)
• The 3 end serves as the starting point for the new
DNA strand

40. • Primase: can start an RNA chain from scratch &
adds RNA nucleotides one at a time using the
parental DNA as a template
Topoisomerase
Primase
RNA
primer
Helicase
Single-strand binding
proteins
5
3
5
53
3

41. Synthesizing a New DNA Strand
• DNA polymerases catalyze the elongation of new
DNA at a replication fork
- most require a primer & a DNA template strand
• The rate of elongation is approx. 500 nucleotides
per second in bacteria & 50 per second in human
cells

42. • Each nucleotide that is added to a growing DNA
strand is a nucleoside triphosphate
• dATP supplies adenine to DNA & is similar to the
ATP of energy metabolism
- the difference is in their sugars: dATP has
deoxyribose while ATP has ribose
• As each dATP joins the DNA strand, it loses 2
phosphate groups as a molecule of pyrophosphate

43. A
C
T
G
G
G
GC
C C
C
C
A
A
A
T
T
New strand 5
end
Template strand 3
end 5 end 3 end
3 end
5 end5 end
3 end
Base
Sugar
Phosphate
Nucleoside
triphosphate
Pyrophosphate
DNA polymerase

44. Antiparallel Elongation
• The double helix has an antiparallel structure
- the 2 strands are oriented in opposite directions
- this affects replication
• DNA polymerases add nucleotides only to the free
3end of a growing strand
- therefore, a new DNA strand can elongate only in
the 5to3direction

45. • Along one template strand of DNA, the DNA
polymerase continuously synthesizes a leading
strand, moving toward the replication fork
Leading strand
Leading strandLagging strand
Lagging strand
Origin of replication
Primer
Overall directions of
replication

46. Origin of replication
RNA primer
Sliding clamp
DNA pol III
Parental DNA
3
5
5
5
5
5
5
3
3
3
Helicase
Single-strand
binding proteins

47. • To elongate the other new strand (the lagging
strand), DNA polymerase must work in the
direction away from the replication fork
• The lagging strand is synthesized as a series of
segments called Okazaki fragments, which are
joined together by DNA ligase

48. Origin of replication
Leading strand
Leading strand
Lagging strand
Lagging strand
Overall directions of
replication
1
2

49. Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
RNA primer
for fragment 2
Okazaki
fragment 2
Overall direction of replication
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
55
5
5
55
5
2
2
2
1
1
1
1
1

51. The DNA Replication Complex
• The proteins that participate in DNA replication
form a large complex called a “DNA replication
machine”
• Recent studies support a model in which DNA
polymerase molecules “reel in” parental DNA &
“extrude” newly made daughter DNA molecules

52. Parental DNA
DNA pol III
Leading strand
Connecting
protein
Helicase
Lagging strandDNA
pol III
Lagging
strand
template
5
5
5
5
5
5
3 3
3
3
3
3

53. Proofreading & Repairing DNA
• DNA polymerases proofread newly made DNA &
replace any incorrect nucleotides
• DNA can be damaged by chemicals, radioactive
emissions, X-rays, UV light, & certain molecules (in
cigarette smoke for example); it can also undergo
spontaneous changes
• Mismatch repair: repair enzymes correct errors in base
pairing
• Nucleotide excision repair: a nuclease cuts out &
replaces damaged stretches of DNA

54. Nuclease
DNA
polymerase
DNA ligase
Nucleotide
Excision Repair
Nuclease

55. Evolutionary Significance of Altered
DNA Nucleotides
• Error rate after proofreading repair is low but not zero
• Sequence changes may become permanent & can be
passed on to the next generation
• These changes (mutations) are the source of the
genetic variation upon which natural selection
operates

56. Replicating the Ends of DNA
Molecules
• Limitations of DNA polymerase create problems for
the linear DNA of eukaryotic chromosomes
• The usual replication machinery provides no way to
complete the 5 ends
- repeated rounds of replication produce shorter
DNA molecules with uneven ends

57. Ends of parental
DNA strands
Leading strand
Lagging strand
Last fragment Next-to-last fragment
Lagging strand RNA primer
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
Second round
of replication
Further rounds
of replication
New leading strand
New lagging strand
Shorter and shorter daughter molecules
3
3
3
3
3
5
5
5
5
5

58. Telomeres
• Nucleotide sequences at the ends of eukaryotic
chromosomal DNA molecules
• Telomeres do not prevent the shortening of DNA
molecules, but they do postpone the erosion of
genes near the ends of DNA molecules
- the shortening of telomeres is thought to be
connected to aging

59. 1 µm

60. • If chromosomes of germ cells became shorter in
every cell cycle, essential genes would eventually be
missing from the gametes they produce
• Telomerase: catalyzes the lengthening of telomeres
in germ cells

61. • The shortening of telomeres might protect cells
from cancerous growth by limiting the number of
cell divisions
• There is evidence of telomerase activity in cancer
cells, which may allow cancer cells to persist

62. • The prokaryotic chromosome is a double-stranded,
circular DNA molecule associated with a small
amount of protein
- the DNA is “supercoiled” & found in the nucleoid
region of the cell
• Eukaryotic chromosomes have linear DNA molecules
associated with a large amount of protein

63. • Chromatin: a complex of DNA & protein found in the
nucleus of eukaryotic cells
• Chromosomes fit into the nucleus through an
elaborate, multilevel system of packing
• Histones: proteins responsible for the 1st level of
DNA packing in chromatin

64. • 10-nm fiber (diameter) – “thin” fiber
– DNA winds around histones to form strings of
nucleosome “beads”
• 30-nm fiber (diameter) – “thick” fiber
– interactions between nucleosomes cause the thin
fiber to coil or fold into this thicker fiber

65. DNA double helix
(2 nm diameter)
Nucleosome
(10 nm “thin” fiber)
Histones
Histone tail
H1
Nucleosomes, or “beads on
a string” (10 nm fiber)

66. 30 nm fiber
Chromatid
(700 nm)
Loops Scaffold
300 nm fiber
Replicated
chromosome
(1,400 nm)30 nm “thick”
fiber
Looped domains
(300 nm fiber)
Metaphase
chromosome

67. • Most chromatin is loosely packed euchromatin in
the nucleus during interphase & condenses prior
to mitosis
• During interphase a few regions of chromatin
(centromeres & telomeres) are highly condensed
into heterochromatin
- this dense packing of chromatin makes it
difficult for the cell to express genetic
information coded in these regions
• Though interphase chromosomes are not highly
condensed, they still occupy specific restricted
regions in the nucleus

68. 5m