Download Molecular Basis of Inheritance

Molecular Basis
Of Inheritance
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
1999 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 Bacteria
Two Strains Of Streptococcus
Rough Strain
(Harmless)
Capsules
Smooth Strain
(Virulent)
©1999 Timothy G. Standish
Transformation Of Bacteria
The Griffith Experiment
OUCH!
+ Control
- Control
- Control
Experimental
©1999 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
things: Protein and DNA
OH
H
H2N C C
CH2
CH2
S
CH3
H
O
H2N C C
OH
Methionine
CH2
SH
O
HO P
NH2
O
O
OH
Cysteine
Some amino acids
contain sulfur, thus
proteins contain sulfur,
but not phosphorous.
OH
H
Nucleotides contain phosphorous,
thus DNA contains phosphorous,
but not sulfur.
©1999 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 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
The Cell Life Cycle
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
©1999 Timothy G. Standish
DNA Replication:
How We Know
There are three ways in which DNA could be replicated:
Conservative - 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
+
Dispersive - In which
sections of the old strands
are dispersed in the new
strands
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
+
+
©1999 Timothy G. Standish
The Meselson-Stahl
Experiment
The Meselson-Stahl experiment demonstrated
HO
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
P
NH2
O
N
O
N
OH
N
N
H
©1999 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,
buy deduction, the
semi-conservative
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
©1999 Timothy G. Standish
Stages 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 proof reading of the DNA to ensure that
mutations are not incorporated into the newly
formed strands of DNA
©1999 Timothy G. Standish
Tools 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 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
Initiation
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
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’
©1999 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’
©1999 Timothy G. Standish
Extension - The Replication Fork
5’
3’
3’
5’
3’
5’
5’
3’
5’
Primase
- Makes
RNA
primers
Laging Strand
Okazaki
fragment
5’
RNA
Primers
3’
5’
Single strand
binding
proteins Prevent DNA
from reanealing
DNA
Polymerase
5’
3’
Helicase Melts DNA
Leading Strand
5’
3’
©1999 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.
©1999 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’
©1999 Timothy G. Standish
Mutation
Excision Repair
5’
3’
3’
5’
5’
3’
3’
5’
3’
EndoNuclease
5’
Nicks
DNA
Ligase
Pol.
3’
5’
©1999 Timothy G. Standish
©1999 Timothy G. Standish
Question:
Problem 1
– 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 %
©1999 Timothy G. Standish
Problem 2
Question:
– Given the following sequence of one strand of DNA,
write out the complimentary strand.
–5’AATACGCGATGCTGGTATC3’
Answer:
–5’AATACGCGATGCTGGTATC3’
–3’TTATGCGCTACGACCATAG5’
©1999 Timothy G. Standish