Download Molecular Basis of Inheritance

Isaiah 33:22
22 For the Lord is our
judge, the Lord is our
lawgiver, the Lord is our
king; he will save us.
©2000 Timothy G. Standish
Molecular Basis
Of Inheritance
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
Outline
1 How we know DNA is the
genetic material
2 When DNA is replicated
3 How DNA is replicated
4 How DNA is maintained
©2000 Timothy G. Standish
Transformation Of Bacteria
Two Strains Of Streptococcus
Rough Strain
(Harmless)
Capsules
Smooth Strain
(Virulent)
©2000 Timothy G. Standish
Transformation Of Bacteria
The Griffith Experiment
OUCH!
+ Control
- Control
- Control
Experimental
©2000 Timothy G. Standish
Avery, MacLeod and McCarty
1944 Avery, MacLeod and McCarty repeated
Griffith’s 1928 experiment with modifications
designed to discover the “transforming factor”
 Extracts from heat-killed cells were digested
with hydrolytic enzymes specific for different
classes of macromolecules:

Enzyme
Transformation?
Lipase
Yes
Protease
Yes
Saccharase
Yes
Nuclease
No
©2000 Timothy G. Standish
The Hershey-Chase
Experiement
The Hershey-Chase experiment showed
definitively that DNA is the genetic material
 Hershey and Chase took advantage of the fact
that T2 phage is made of only two classes of
macromolecules: Protein and DNA

H
H2N C C
CH2
CH2
S
CH3
H
O
H2N C C
OH
Methionine
CH2
SH
OH
O
OH
Cysteine
Some amino acids
contain sulfur, thus
proteins contain sulfur,
but not phosphorous.
HO P
NH2
O
O
OH
H
Nucleotides contain phosphorous,
thus DNA contains phosphorous,
but not sulfur.
©2000 Timothy G. Standish
S35
T2 grown in
containing media
incorporate S35
into their proteins
Using S35 Bacteria grown in
T2 attach to bacteria and
inject genetic material
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The supernatant is
radioactive, but the
pellet is not.
Blending causes phage
protein coat to fall off
Did protein enter the bacteria?
Is protein the genetic material?
P32
T2 grown in
containing media
incorporate P32
into their DNA
Using P32 Bacteria grown in
T2 attach to bacteria and
inject genetic material
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The pellet is
radioactive, but the
supernatant is not.
Blending causes phage
protein coat to fall off
Did DNA enter the bacteria?
Is DNA the genetic material?
When DNA Replication
Occurs
Typically DNA replication only occurs when cells
are preparing to divide (there are some
exceptions)
 The cell lifecycle is well defined and can be
divided into four stages:

– Gap 1 (G1) - The growth phase in which most cells are
found most of the time
– Synthesis (S) - During which new DNA is synthesized
– Gap 2 (G2) - The period during which no transcription
or translation occurs and final preparations for division
are made
– Mitosis - Cell division
©2000 Timothy G. Standish
The Cell Lifecycle
Gap 1 - Doubling
of cell size.
Regular cellular
activities.
Transcription and
translation etc.
Synthesis of DNA Regular cell
activities cease and
a copy of all nuclear
DNA is made
S
G1
G2
M
Gap 2 - Final
preparation for
division
Mitosis - Cell
division
©2000 Timothy G. Standish
A Nucleotide
Adenosine Mono Phosphate (AMP)
Phosphate
HO
H+
Nucleotide
OH
P
O
Base
N
H
O
5’CH2
4’
NH2
H
N
O
1’
Sugar
3’
OH
2’
H
OH
N
N
Nucleoside
Purines
NH2
Adenine
N
N
N
O
CH3
(DNA)
N
Guanine
NH
N
Thymine
O
NH2
Uracil
(RNA)
NH
N
O
N
N
Pyrimidines
NH
O
N
O
NH2
Cytosine
N
N
O
Base Pairing
Guanine And Cytosine
-
+
+
+
-
Base Pairing
Adenine And Thymine
+ -
Adenine
-
+
Thymine
Base Pairing
Adenine And Cytosine
+
-
-
Base Pairing
Guanine And Thymine
+
+
P
HO
NH2
O
N
O
CH2
OH
N
N
O
H
N
O
CH2
O
HO
P
O
O
N
O
CH2
OH
H
H2O
NH
N
O
HO
P
O
H
O
NH2
N
O
CH2
O
H
O
H
H2O
N
O
CH2
N
O
O
O
CH2
O
P
HO
H
O
OH
HO
P
NH2
HO
P
O
H
O
HO
O
D
N
A
OH
-
-
-
-
-
-
G
-
3.4 nm
1 nm
-
-
Minor
groove
C
G C
T A
A T
-
The Watson - Crick
Model Of DNA
G C
T A
C G
A T
Major
groove
A T
C G
G C
0.34 nm
T A
-
-
-
-
-
-
-
-
-
-
-
©2000 Timothy G. Standish
-
DNA Replication:
How We Know
 There are three
Conservative - Old
ways in which DNA could be replicated:
Semiconservative Old strands serve as
templates for new
strands resulting in
double-stranded
DNA made of both
old and new strands
double-stranded
DNA serves as a
template for two
new strands which
then join together,
giving two old
strands together
and two new
Dispersive - In
strands together
which sections of
the old strands are
dispersed in the
new strands
+
+
+
©2000 Timothy G. Standish
The Meselson-Stahl
Experiment
OH
N
The Meselson-Stahl experiment
N
N
N N
demonstrated that replication is
semiconservative
OH H
 This experiment took advantage of
the fact that nucleotide bases contain nitrogen
 Thus DNA contains nitrogen
 The most common form of Nitrogen is N14 with 7
protons and 7 neutrons
 N15 is called “heavy nitrogen” as it has 8 neutrons
thus increasing its mass by 1 atomic mass unit

HO P
O
H2
O
©2000 Timothy G. Standish
The Meselson-Stahl
Experiment
Transfer to
normal N14
media
Bacteria grown in
N15 media for
several replications
The conservative and
dispersive models
make predictions that
do not come true thus,
by deduction, the semiconservative model
must be true.
After 20 min.
(1 replication)
transfer DNA
to centrifuge
tube and
centrifuge
Prediction after
2 or more
replications
X
X X
©2000 Timothy G. Standish
Stages of Replication
Replication can be divided into three stages:
1 Initiation - When DNA is initially split into two
strands and polymerization of new DNA is started
2 Elongation - When DNA is polymerized
3 Termination - When the new strands of DNA are
completed and some finishing touches may be put
on the DNA
 Both elongation and termination may involve
proofreading of the DNA ensuring that mutations
are not incorporated into newly formed DNA
strands

©2000 Timothy G. Standish
Tools of Replication
 Enzymes
are the tools of replication:
 DNA Polymerase - Matches the
correct nucleotides then joins adjacent
nucleotides to each other
 Primase - Provides an RNA primer to
start polymerization
 Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
©2000 Timothy G. Standish
More Tools of Replication
 Helicase
- Unwinds the DNA and melts it
 Single-Strand Binding Proteins - Keep
the DNA single stranded after it has been
melted by helicase
 Topisomerase - Relieves torsional strain
in the DNA molecule
 Telomerase - Finishes off the ends of
DNA strands
©2000 Timothy G. Standish
Initiation
 Initiation
starts at specific DNA sequences
called origins (Ori C = origin in E. coli
chromosomes)
 Long linear chromosomes have many origins
 First the origin melts (splits into two single
strands of DNA)
 Next primers are added
 Finally DNA polymerase recognizes the
primers and starts to polymerize DNA 5’ to 3’
away from the primers
©2000 Timothy G. Standish
Initiation - Forming the
Replication Eye
Origin of Replication
5’
3’
3’
5’
3’
5’
5’
3’
3’
5’
5’
5’
3’
3’
5’
3’
3’
5’
5’
3’
©2000 Timothy G. Standish
Large Linear Chromosomes Have
Many Origins Of Replication
Origins of Replication
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
©2000 Timothy G. Standish
Extension - The Replication Fork
5’
3’
3’
5’
3’
5’
5’
3’
5’
Primase
- Makes
RNA
primers
Lagging Strand
Okazaki
fragment
5’
RNA
Primers
3’
5’
Single-strand
binding
proteins Prevent DNA
from reannealing
DNA
Polymerase
5’
3’
Helicase Melts DNA
Leading Strand
5’
3’
©2000 Timothy G. Standish
Extension - Okazaki Fragments
5’
3’
Okazaki Fragment
DNA
Pol.
3’
5’
RNA Primer
DNA Polymerase has 5’ to 3’ exonuclease activity.
When it sees an RNA/DNA hybrid, it chops out the
RNA and some DNA in the 5’ to 3’ direction.
5’
3’
DNA
Pol.
RNA and DNA Fragments
3’
5’
RNA Primer
DNA Polymerase falls off leaving a nick.
5’
3’
Ligase
3’
5’
RNA Primer
Nick
The nick is removed when
DNA ligase joins (ligates) the
DNA fragments.
©2000 Timothy G. Standish
Mutation
When Mistakes Are Made
5’
DNA
Pol.
5’
5’
DNA
Pol.
3’ to 5’ Exonuclease activity
5’
3’
DNA
Pol.
3’
5’
3’
5’
©2000 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
EndoNuclease
5’
©2000 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
5’
3’
EndoNuclease
5’
Nicks
DNA
Pol.
3’
5’
©2000 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
5’
3’
EndoNuclease
5’
DNA
Pol.
3’
5’
©2000 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
EndoNuclease
5’
Nicks
5’
3’
Ligase
3’
5’
Nick
©2000 Timothy G. Standish
©2000 Timothy G. Standish

Question:
Problem 1
– If an organism’s DNA is 32 % adenine, what percent
guanine, thymine, and cytosine are found in the DNA?

Answer:
– As adenine always pairs with thymine, there must be
32 % thymine
– % GC = 100 % - (T% + A%) = 100 % - (32 % + 32
%) = 36 %
– The proportion of guanine to cytosine has to be equal
as they pair with one another thus G and C % = 36 % /
2 = 18 %
– G = 18 %, T = 32 % and C = 18 %
©2000 Timothy G. Standish
Problem 2

Question:
– Given the following sequence of one strand of DNA,
write out the complementary strand.
–5’AATACGCGATGCTGGTATC3’

Answer:
–5’AATACGCGATGCTGGTATC3’
–3’TTATGCGCTACGACCATAG5’
©2000 Timothy G. Standish