©1999 Timothy G. Standish
Molecular Basis Molecular Basis Of InheritanceOf Inheritance
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
1999 Ig Nobel Prizes1999 Ig Nobel Prizes Science Education. The Kansas Board of Education and the Colorado State Board of
Education, for mandating that children should not believe in Darwin's theory of evolution.
Medicine. Arvid Vatle of Norway, for carefully collecting, classifying, and contemplating which kinds of containers his patients chose when submitting urine samples.
Chemistry. Takeshi Makino, of the Safety Detective Agency in Japan, for his involvement with an infidelity detection spray that wives can apply to their husbands' underwear.
Biology. Paul Bosland, of the New Mexico State University's Chile Pepper Institute, for breeding a spiceless jalapeno chile pepper.
Managed Health Care. The late George and Charlotte Blonsky of New York City and San Jose, California, for inventing a childbirth device in which the woman is strapped to a circular table that is then rotated at high speed.
©1999 Timothy G. Standish
Transformation Of BacteriaTransformation Of BacteriaTwo Strains Of Two Strains Of StreptococcusStreptococcus
Capsules
Smooth Strain(Virulent)
Rough Strain(Harmless)
©1999 Timothy G. Standish
Experimental
Transformation Of BacteriaTransformation Of BacteriaThe Griffith ExperimentThe Griffith Experiment
- Control
+ Control
- Control
OUCH!
©1999 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 things: 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?
©1999 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 life cycle 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
©1999 Timothy G. Standish
G1G1
MM
G2G2
SS
The Cell Life CycleThe Cell Life CycleGap 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
©1999 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 - The old double-stranded DNA serves as a template for two new strands that are then joined together, thus giving two old strands together and two new strands together
Old
Semi-conservative - In which old strands serve as templates for new strands resulting in double-stranded DNA made up of an old and new strand
Old
Dispersive - In which sections of the old strands are dispersed in the new strands
©1999 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 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
OH
HOH
P
O
HO ONH2
N N
N N
©1999 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
Semi-c
onservativ
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.
©1999 Timothy G. Standish
Stages of ReplicatonStages of Replicaton Replication can be divided into three stages: Initiation - When DNA is initially split into two strands and
polymerization of new DNA is started Elongation - When DNA is polymerized 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 some
proofreading of the DNA to ensure that mutations are not incorporated into the newly formed strands of DNA
©1999 Timothy G. Standish
Tools of ReplicationTools of Replication There are three major enzymes involved in
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”)
©1999 Timothy G. Standish
More Tools of ReplicationMore 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
©1999 Timothy G. Standish
InitiationInitiation Initiation starts at specific DNA sequences called origins
(Ori C = origin in E. coli chromosomes) Large linear chromosomes may 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
©1999 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’
©1999 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
©1999 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
©1999 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
©1999 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
©1999 Timothy G. Standish
Thim
ine
Dimer
5’ 3’
3’ 5’
MutationMutationExcision RepairExcision Repair
3’
5’ 3’
5’
5’ 3’
3’ 5’
DNAPol.
DNAPol.
Ligase
Endo-Nuclease
Ligase
Nicks
©1999 Timothy G. Standish
Problem 1Problem 1 Question:
– If an organisms 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 %