Download Chapter 16:

Chapter 16:
The Molecular Basis of Inheritance
• Mendel discovered the existence of
heritable factors…
• Just about 60 years ago biologists had a
dilemma: Where were the genes located?
• T.H. Morgan’s group showed that genes
are located on chromosomes.
• This led to the next question: Which one,
protein or DNA, is the “genetic material”?
“Little was known abut nucleic acids, whose
physical and chemical properties seemed far
too uniform to account for the multitude of
specific inherited traits exhibited by every
organism.”
Appropriate experimental organisms:
Viruses and Bacteria
Scientists Who Contributed to the
Discovery of DNA as The Unit of Heredity:
1)
2)
3)
4)
5)
6)
Frederick Griffith
Avery, MacLeod & McCarty
Hershey and Chase
Erwin Chargaff
Rosiland Franklin
James Watson and Francis Crick
1) Frederick Griffith 1928
Transformation, the change in a genotype and phenotype due
to the assimilation of external material by a cell, is possible.
• Studied Streptococcus pneumoniae, bacterium
• 2 varieties: pathogenic S (smooth) or harmless R (rough)
• Experiment:
1) kill pathogenic strain with heat (inject in mouse)
2) mix dead pathogenic bacteria with harmless live bacteria.
(then inject in mouse)
Figure 16.1 Transformation of bacteria
•Results: some bacteria became pathogenic
(injected mice with the bacterial strain- they died)
•Transformation, the change in a genotype and phenotype
due to the assimilation of external material by a cell,
is possible.
2) Avery, MacLeod and McCarty
The transforming agent was DNA.
Attempted successfully to pinpoint the
transforming agent.
Experiment:
1. Purified various chemicals from
the heat-killed pathogenic
bacteria.
2. tried to transform live “harmless”
or non-pathogenic bacteria with
each chemical.
• Result: only DNA worked.
• The transforming agent was
DNA.
3) Alfred Hershey & Martha Chase
DNA is a virus’ genetic material
•
•
•
1952 used bacteria infecting viruses
bacteriophages “bacteria eaters” (virus) = DNA + protein
Experiment to show that it was DNA not protein that
actually entered the bacterium- DNA was virus’ genetic
material (viral DNA can program cells).
1. Tag DNA and protein with different radioactive isotopes
then centrifuge cells- DNA is in the pellet, the protein coat
is in the supernatant (liquid above the pellet.)
• Infect E. coli w/ radioactive-sulfur (35S protein) grown
viruses. Centrifuge cells. Radioactivity in supernatant.
• Infect E. coli w/ radioactive-phosphorus (32P DNA) grown
viruses. Centrifuge cells. Radioactivity in pellet.
• Pellet = Cells , supernatant = outside of cells
Bacteriophage reproduction
• DNA enters cells
• Protein remains outside.
• Phages act like a hypodermic needle- inject
DNA into their host cell.
Figure 16.2a The Hershey-Chase experiment: phages
Conclusion:
DNA
(not
protein)
functions as
the T2
phage’s
genetic
material.
4) Erwin Chargaff
in DNA, the nucleotides A & T exist in equal proportions; as do C and G
• 1947 biochemist who analyzed the base composition of
DNA from different organisms.
• DNA composition varies from one species to another:
• In any one species, the amounts of the four nitrogenous
bases are not all equal but are present in a characteristic
ratio.
• A = T (approximately) and G = C Chargaff’s rules
Chargaff’s rules:
A=T
G=C
This is how we know
The base pair rules.
5) Rosiland Franklin
shape of DNA is twisted
• X-ray diffraction/ X-ray crystallography
• Basically took x-ray picture of DNA- based on the
“shadow” it cast.
• Worked down the hall from Watson & Crick, who
were also studying the structure of DNA.
• Victim of sexism… pioneer of her time.
• Nobel Prize can only go to three people… the
structure of DNA is credited to Watson, Crick, &
Wilkins.
Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA
6) James Watson and Francis Crick
DNA is double helix
•
•
•
•
American (Watson) and Englishman (Crick)
Interpret Franklin’s pictures of DNA
DNA helical in shape
Able to calculate width of helix and spacing of
nitrogenous bases along it.
• Double helix
• Explained Chargaff’s rules.
Figure 16.0 Watson and Crick
Figure 16.0x James Watson
•
•
•
•
•
The Molecular
Structure of
DNA
Backbone: sugar-phosphate
In a 3’ to 5’ and 5’ to 3’ direction
Note the numbering of the sugar!
2 types of Nitrogenous Bases:
Purines:
Adenine & Guanine
• Pyrimindines:
Thymine & Cytosine
• Base Pairing: A-T & G-C
G & C look like eachother
Unnumbered Figure (page 292) Purine and pyridimine
1/2 of the DNA Double Helix
1/2 of the Twisted Ladder
1)What are the uprights of the ladder?
2)What are the rungs of the ladder?
3)What would the other side look like?
4)What kinds of bonds hold sugars to
phosphates?
5)What kinds of bonds hold nitrogenous
bases together?
6)Which side runs 3’ to 5’?
1/2 of the DNA Double Helix
1/2 of the Twisted Ladder
1) Uprights = sugar-phosphates
2) Rungs = bases
3) A-T-G-C (inverted sugar-phosphates)
4) Covalent bonds
5) Hydrogen bonds (2 AT & 3 CG)
6) Right side- 5’ = phosphate group
Strands are “anti-parallel”
Figure 16.5 The double helix
Quiz Time
• What holds the two strands of DNA
together?
• HYDROGEN BONDS (2 AT or 3 GC)
• What shape is the DNA molecule?
• DOUBLE HELIX
• When did Watson and Crick complete their
model?
• FEBRUARY 28th 1953 @ 7:07am
Figure 16.8 Three alternative models of DNA replication
Semiconservative Model- two strands of the parental molecule separate,
and each functions as a template for synthesis of a new complementary strand.
DNA REPLICATION
• What is a template? 1/2 DNA Double Helix… a single
strand of DNA.
• What was Watson and Crick’s template theory? DNA is a
pair of templates complementary to each other. Implies
a copying mechanism: each new strand is 1/2 original
template and 1/2 new complementary strand.
• What is DNA polymerase? The enzyme that connects a
nucleoside-tri-phosphate to a DNA template nucleotide
according to base-pair rules.
• Each step of DNA replication is managed by a specific
enzyme.
• What fuels this process? Phosphates from the nucleosidetri-phosphate.
Figure 16.11 Incorporation of a nucleotide into a DNA strand
Figure 16.7 A model for DNA replication: the basic concept (Layer 1)
Figure 16.7 A model for DNA replication: the basic concept (Layer 2)
HELICASE ENZYME splits the DNA double helix
Figure 16.7 A model for DNA replication: the basic concept (Layer 3)
DNA POLYMERASE attracts matching nucleotides and covalently
bonds them to the free 3’ end of the existing strand.
Also “proof-reads” for mistakes.
Figure 16.7 A model for DNA replication: the basic concept (Layer 4)
PROKARYOTIC REPLICATION
• Prokaryotic chromosomes are single, circular,
and about 5 million base pairs.
• Replication begins at the origin of replication.
• The direction of replication is 3’ to 5’ along the
template strand… new DNA is created 5’ to 3’.
• Replication can be as fast as one hour- some
quicker than 20 minutes.
• Ex. A staph infection is deadly because
doubling occurs every 20 minutes.
EUKARYOTIC REPLICATION
• Eukaryotic chromosomes are LINEAR
• Replication begins at the originS of
replication (multiple spots)
• The direction of replication is 3’ to 5’
along the template strand (so new
strand is “elongated” 5’ to 3’)
• Replication can be as fast as a few hours
in humans- even though our 6 billion
base pairs is over a 1000x more DNA
than in bacteria.
• Because eukaryotic chromosomes are
larger, replication begins at many sites
along the giant DNA molecule of each
chromosome.
Figure 16.10 Origins of replication in eukaryotes
Helicase- unzips the DNA at an “origin of replication”
Single Stranded Binding Proteins- keep strands apart
Primase attracts RNA nucleotides to form RNA primer
DNA Polymerase attracts DNA nucleosides to build the new strand
and replaces the primer with DNA.
5. DNA ligase joins Okazaki fragments on the lagging strand
1.
2.
3.
4.
LEADING STRAND is built by the continuous
addition of nucleotides along the 3’-5’template
toward the replication fork. Elongation occurs
5’ to 3’ because DNA polymerase can only bond
Nucleotides to the 3’ end of a nucleotide.
LAGGING STRAND is built by the fragmented addition of nucleotides
along the 5’-3’ template strand away from the replication fork. Since
polymerase attaches to the template on the 3’ end and moves toward
the 5’ end, elongation occurs in “spurts” & creates Okazaki fragments.
• DNA strands are anti-parallel
5’------------------------------------------3’
3’------------------------------------------5’
• 5’ end is the phosphate end
• 3’ end is the hydroxyl end.
• DNA POLYMERASE attaches to the
template on the 3’ end and moves
toward the 5’ end.
• ELONGATION of the new strand
occurs in the 5’ to 3’ direction
because DNA polymerase can only
add nucleotides to the free 3’ end of
an existing nucleotide… this is why
PRIMER must be laid down first.
1) getting started: origins of replication
•
•
•
At the origin(s) of replication,
proteins attach to the DNA
separating the two strands with
HELICASE and holding them
apart with SINGLE
STRANDED BINDING
PROTEINS opening up one or
many “replication bubble (s)”.
At each end of a replication
bubble is a replication fork a
Y-shaped region where the new
strands of DNA are elongating.
Replication proceeds in both
directions along the template
from it’s 3’ to 5’ end.
Figure 16.14 Priming DNA synthesis with RNA
2)Elongating a new DNA strand:
•
•
•
•
•
PROBLEM!!! DNA polymerase can only
add to a started chain… so how do we
start the new chain?
PRIMASE adds RNA nucleotides to
create the beginning of the complementary
new strand = PRIMER (a stretch of about
10 nucleotides in Eukaryotes)
Next, elongation of new DNA at a
replication fork is catalyzed by enzymes
called DNA POLYMERASES.
DNA POLYMERASE covalently bonds
the phosphate of the new nucleotide to the
3’ sugar of the next nucleotide.
A different DNA polymerase switches
DNA nucleotides for the primer.
HOW LONG DOES IT TAKE?
• 500 nucleotides /second in bacterial cells
• 50/second in human cells
• SOURCE OF ENERGY??? They are
actually A,T, G, C nucleoside triphosphates not nucleotides.
Figure 16.13 Synthesis of leading and lagging strands during DNA replication
REPLICATION PROBLEM:
telomeres
Telomeres are the ends of linear
Chromosomes.
There is a problem with DNA
replication where the
daughter molecules of DNA
get shorter and shorter each time
the DNA is copied.
Some cells are “immortal” and
carry an enzyme called
TELOMERASE which solves
the “end replication problem”.
Figure 16.19b Telomeres and telomerase
The 5’ end of the
Dna has a problem
Being replicated.
DNA can then get
Shorter and shorter
With each reproduction.
Enzyme- telomerase
Extends the 3’ end of the
DNA and then the other
End is extended with primase
DNA polymerase, and ligase.
Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes
ORGANELLE REPLICATION
• The two organelles that have DNA are the
MITOCHONDRION & CHLOROPLAST
• Their chromosomes are CIRCULAR, JUST
LIKE A PROKARYOTE.
• These organelles are inherited from an
organism’s MOTHER (mitochondrial
disorders pose a problem)
* Relate this to our family tree… Eve.
Mutations are changes in the DNA code.
1) When chromosomes replicate, the error rate is:
1/10,000 base pairs or 1/1billion
To correct these errors:
DNA polymerase itself proofreads each nucleotide
against its template (like “delete” key) & mismatch
repair enzymes fix missed errors (NUCLEASE).
2) Additional factors that cause mutation: mutagens,
carcinogens
a. Chemicals
b. X and UV radiation- ex. thymine dimer
(bond to e/o not the A’s fixed by excision repair)
c. Bases undergo spontaneous changes
Figure 16.17 Nucleotide excision repair of DNA damage
DNA REPLICATION
EUKARYOTIC
DNA REPLICATION
•
•
•
•
•
•
•
•
•
•
•
•
•
Origin of Replication
Helicase
Replication fork
Single strand binding
proteins
Primase
Primer
DNA polymerase
Direction 3’ --> 5’
Elongation 5’ --> 3’
Leading strand
Lagging strand
Okazaki fragments
DNA ligase
Leading Strand (narrated)
DNA REPLICATION narrated