©2000 Timothy G. Standish Isaiah 33:22 22For the Lord is our judge, the Lord is our lawgiver, the...
-
Upload
jane-gilbert -
Category
Documents
-
view
217 -
download
1
Transcript of ©2000 Timothy G. Standish Isaiah 33:22 22For the Lord is our judge, the Lord is our lawgiver, the...
©2000 Timothy G. Standish
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 Molecular Basis Of InheritanceOf Inheritance
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
OutlineOutline
1How we know DNA is the genetic material
2When DNA is replicated
3How DNA is replicated
4How DNA is maintained
©2000 Timothy G. Standish
Transformation Of BacteriaTransformation Of BacteriaTwo Strains Of Two Strains Of StreptococcusStreptococcus
Capsules
Smooth Strain(Virulent)
Rough Strain(Harmless)
©2000 Timothy G. Standish
Experimental
Transformation Of BacteriaTransformation Of BacteriaThe Griffith ExperimentThe Griffith Experiment
- Control
+ Control
- Control
OUCH!
©2000 Timothy G. Standish
Avery, MacLeod and McCartyAvery, 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:
NoNuclease
YesProtease
YesLipase
Transformation?Enzyme
YesSaccharase
©2000 Timothy G. Standish
The Hershey-Chase The Hershey-Chase ExperiementExperiement
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
HOH
P
O
OH
HO ONH2
Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur.
H
OH
OH2N CC
CH2
SH
H
OH
OH2N C
CH3
C
CH2
CH2
S Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous.
CysteineMethionine
Using SUsing S3535Bacteria grown in normal non-radioactive media
T2 grown in S35 containing media incorporate S35 into their proteins
Blending causes phage protein coat to fall off
T2 attach to bacteria and inject genetic material
Is protein the genetic material?
When centrifuged, phage protein coats remain in the supernatant while bacteria form a pelletThe supernatant is radioactive, but the pellet is not.
Did protein enter the bacteria?
Using PUsing P3232Bacteria grown in normal non-radioactive media
T2 grown in P32 containing media incorporate P32 into their DNA
Blending causes phage protein coat to fall off
T2 attach to bacteria and inject genetic material
Is DNA the genetic material?
When centrifuged, phage protein coats remain in the supernatant while bacteria form a pelletThe pellet is radioactive, but the supernatant is not.
Did DNA enter the bacteria?
©2000 Timothy G. Standish
When DNA Replication When DNA Replication OccursOccurs
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
G1G1
MM
G2G2
SS
The Cell LifecycleThe Cell LifecycleGap 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
Gap 2 - Final preparation for division
Mitosis - Cell division
OH
OCH2
Sugar
H
HH
A NucleotideA NucleotideAdenosine Mono Phosphate (AMP)Adenosine Mono Phosphate (AMP)
OH
NH2
N
N N
N
BaseP
O
OH
HO O
Phosphate
2’3’
4’
5’
1’Nucleotide
Nucleoside
H+
-
Pyrimidines
NH2
O
N
N NH
N
Guanine
N
N
Adenine
N
N
NH2
N O
NH2
N O
NH2
NCytosine
Uracil(RNA)CH3
N ON
O
NH
N ON
O
NH
Thymine(DNA)
Purines
NO
H
NO
N
NH C
ytosine
H
O
NN
N
N
N
H
H
Guanine -+
+
+
-
-
Base PairingBase PairingGuanine And CytosineGuanine And Cytosine
CH 3
N
O
N
ON
H+
- ThymineN
NN
N
HN H
-
+Adenine
Base PairingBase PairingAdenine And ThymineAdenine And Thymine
Base PairingBase PairingAdenine And CytosineAdenine And Cytosine
NO
H
NO
N
NH C
ytosine-
+
-
N
NN
N
HN
H
-
+
Adenine
Base PairingBase PairingGuanine And ThymineGuanine And Thymine
CH
3
NO
N
O
NH+
- Thymine
H
O
NN
N
N
N
H
H
Guanine
+
+
-
SU
GA
R-P
HO
SP
HA
TE
BA
CK
BO
NE
H
P
O
HO
O
O
CH2
HOH
P
O
O
HO
O
O
CH2
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
B A
S E
S
DDNNAA
OH
P
O
HO
O
O
CH2
HO
O
H 2N
NHN N
N H
H
P HO
O
O
CH2
OO
N
O
H 2N
NH
H2O
H OH
P
O
HO
O
O
CH2
CH 3
O
O
HNN
H2O
©2000 Timothy G. Standish
The Watson - Crick The Watson - Crick Model Of DNAModel Of DNA
3.4 nm1 nm
0.34 nm
Majorgroove
Minorgroove
A T
T AG C
C G
C GG C
T A
A T
G CT A
A TC G
--
-
-
---
--
--
--
-
--
--
-
---
--
--
--
-
-
©2000 Timothy G. Standish
DNA Replication:DNA Replication:How We KnowHow We Know
There are three ways in which DNA could be replicated:
+
NewOld
+
Old
N
ewOld
N
ew
+
Old +
N
ewOld +
N
ew
OldConservative - Old double-stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together
OldSemiconservative - Old strands serve as templates for new strands resulting in double-stranded DNA made of both old and new strands
Old
Dispersive - In which sections of the old strands are dispersed in the new strands
©2000 Timothy G. Standish
The Meselson-Stahl The Meselson-Stahl ExperimentExperiment
The Meselson-Stahl experiment demonstrated that replication is semiconservative
This experiment took advantage of the fact that nucleotide bases contain nitrogen
Thus DNA contains nitrogen
OH
HOH
P
O
HO ONH2
N N
N N
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
©2000 Timothy G. Standish
After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge
Disper
sive m
odel
predict
ion
Conservativ
e
model pre
diction
Semico
nservativ
e
model pre
diction
The Meselson-Stahl The Meselson-Stahl ExperimentExperiment
Prediction after 2 or more replications
Bacteria grown in N15 media for several replications
Transfer to normal N14 media
X
X
XThe conservative and dispersive models make predictions that do not come true thus, by deduction, the semi-conservative model must be true.
©2000 Timothy G. Standish
Stages of ReplicationStages 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 ReplicationTools of ReplicationEnzymes 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 ReplicationMore Tools of Replication
Helicase - Unwinds the DNA and melts itSingle-Strand Binding Proteins - Keep the DNA
single stranded after it has been melted by helicaseTopisomerase - Relieves torsional strain in the
DNA moleculeTelomerase - Finishes off the ends of DNA
strands
©2000 Timothy G. Standish
InitiationInitiation 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 Initiation - Forming the Replication EyeReplication Eye
3’ 5’
3’5’5’
5’
3’
3’
Origin of Replication
5’
3’
3’
5’
5’3’
5’
5’
5’
3’
3’3’
©2000 Timothy G. Standish
Large Linear Chromosomes Have Large Linear Chromosomes Have Many Origins Of ReplicationMany Origins Of Replication
5’3’
3’5’
5’3’
3’5’
3’5’
5’3’
3’5’
5’3’
3’5’
5’3’
Origins of Replication
©2000 Timothy G. Standish
Leading StrandLeading Strand
Lagging StrandLagging Strand
3’
5’3’
5’
Extension - The Replication ForkExtension - The Replication Fork5’
5’5’3’
3’
5’3’3’
5’
Single-strand binding proteins - Prevent DNA from re-annealing
DNA Polymerase
Okazaki fragment
RNA Primers
Primase - Makes RNA primers
5’3’
5’
Helicase - Melts DNA
©2000 Timothy G. Standish
Extension - Okazaki FragmentsExtension - Okazaki Fragments
The nick is removed when DNA ligase joins (ligates) the DNA fragments.
3’ 5’5’ 3’
RNA PrimerOkazaki Fragment
3’ 5’5’ 3’
RNA PrimerRNA and DNA Fragments
Nick
3’ 5’5’ 3’
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.
DNA Polymerase falls off leaving a nick.
DNAPol.
DNAPol.
Ligase
©2000 Timothy G. Standish
MutationMutationWhen Mistakes Are MadeWhen Mistakes Are Made
5’ 3’
5’
DNAPol.
5’
5’ 3’
5’ 3’
5’
DNAPol.
DNAPol.
Mism
atch
3’ to 5’ Exonuclease activity
©2000 Timothy G. Standish
Thim
ine
Dimer
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
5’ 3’
3’ 5’
Endo-Nuclease
©2000 Timothy G. Standish
5’ 3’
3’ 5’
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
Endo-Nuclease
NicksDNAPol.
©2000 Timothy G. Standish
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
5’ 3’
3’ 5’
DNAPol.
Endo-Nuclease
©2000 Timothy G. Standish
5’ 3’
3’ 5’
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
DNAPol.
Ligase
Endo-Nuclease
Nicks
Nick
Ligase
©2000 Timothy G. Standish
©2000 Timothy G. Standish
Problem 1Problem 1 Question:
– 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 2Problem 2 Question:
– Given the following sequence of one strand of DNA, write out the complementary strand.
–5’AATACGCGATGCTGGTATC3’ Answer:
–5’AATACGCGATGCTGGTATC3’–3’TTATGCGCTACGACCATAG5’