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THE CENTRAL DOGMA
Objectives
Describe how Griffith’s discovery of “transformation”
suggested DNA, not proteins, were the genetic material
Understand how Harvey and Chase proved DNA was
the genetic material
Describe the structure and function of DNA
Make a complimentary strand of DNA
Central Dogma
DNA
Replication
&
Repair
DNA
Structure
&
Function
DNA discovery timeline
1868 Johann Miescher
Purify the contents of the nucleus: an organic acid with phosphate
1928 Frederick Griffith
Hereditary material not killed by heat
Hereditary material can transform bacteria
1900s Oswald Avery
DNA, not protein, causes transformations
1950s Harvey and Chase
DNA, not protein, contains the genetic material of bacteriophages
1949 Erwin Chargaff
Amount of adenine = amount of thymine, guanine = cytosine
1900s Rosalind Franklin
DNA has more than one chain
DNA is arranged in a helix
Discovered the length and diameter of DNA
1953 James Watson and Francis Crick
Build a model of the structure of DNA
Griffith Discovers Transformation
1928
He was attempting to develop a vaccine
Isolated two strains of Streptococcus pneumoniae
Rough strain was harmless
Smooth strain was pathogenic
Griffith Discovers Transformation
4. Mice injected with
live R cells plus heat-
killed S cells
Mice die. Live S cells in
their blood
1. Mice injected with
live cells of harmless
strain R
Mice live. No live R
cells in their blood
2. Mice injected with live
cells of killer strain S
Mice die. Live S cells in
their blood
3. Mice injected with
heat-killed S cells
Mice live. No live S cells
in their blood
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Transformation
What happened in the fourth experiment?
The harmless R cells had been transformed by
material from the dead S cells
Descendents of the transformed cells were also
pathogenic
The question is: what was the material???
What Is the
Transforming Material?
Avery found that cell extracts treated with
protein-digesting enzymes could still transform
bacteria
Cell extracts treated with DNA-digesting
enzymes lost their transforming ability
Concluded that DNA, not protein, transforms
bacteria
DNA
inside
protein
coat
Hollow
sheath
Tail
fiber
A bacteriophage
is a virus that infects bacteria
Question: what
infects the
bacterium, the
protein or the
DNA?
Virus particle
labeled with
35
S
Harvey and Chase
show protein does
not infect the
bacterium
DNA being
injected into
bacterium
35
S remains
outside cells
DNA being injected
into bacterium
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P remains inside cells
virus particle
labeled with
32
P
Harvey and Chase
show DNA infects the
bacterium
Structure of Nucleotides
in DNA
Each nucleotide consists of
Deoxyribose (5-carbon sugar)
Phosphate group
A nitrogen-containing base
There are four bases:
Adenine, Guanine, Thymine, Cytosine
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Nucleotide Bases
phosphate
group
deoxyribose
ADENINE
(A)
THYMINE
(T)
CYTOSINE
(C)
GUANINE
(G)
Rosalind Franklin:
discovery of DNA structure
Credit: © Science VU/LBL/Visuals Unlimited
X-ray diffraction
James Watson
Francis Crick
1962 Nobel Prize
DNA
structure
Two strands
Of nucleotides
Held together by
hydrogen bonds
Coiled in a double
helix
Quick Quiz Question
What is the complementary DNA strand
to:
TACGGCCTTAAGACG
Objectives
Describe the steps and enzymes involved in
DNA replication
Define “cloning”
Explain why Dolly the sheep was such a big
breakthrough
Why don’t clones (or identical twins) look
identical?
Explain why new research is not using egg cells.
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DNA
Replication
new new old old
Hydrogen bonds
between strands are
easily broken
Each parent strand
remains intact
Every DNA molecule is
half “old” and half “new”
Base Pairing
during Replication
Each old strand
serves as the
template for
complementary
new strand
Enzymes in Replication
Enzymes unwind the two strands and their
complementary base pairs unzip
DNA polymerase attaches new complementary
nucleotides
DNA ligase fills in gaps
Enzymes wind two strands together
DNA Repair
Mistakes can occur during replication: these
mistakes are called mutations
DNA polymerase can read correct sequence
from complementary strand and, together with
DNA ligase, can repair mistakes in incorrect
strand
Cloning
Making a genetically identical copy of an
individual
Researchers have been creating clones for
decades
These clones were created by embryo splitting,
a.k.a., artificial twinning
Showed that differentiated cells could be
used to create clones
Sheep udder cell was combined with
enucleated egg cell
Dolly is genetically identical to the sheep that
donated the udder cell
Dolly:
Cloned from an Adult Cell
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Cloning
Body cell
Egg cell Remove nucleus
Remove nucleus
Add body cell
nucleus to egg
cell
Trigger
cell
division
More Clones
Numerous species have now been cloned
Mice, pigs, cattle, cats, etc.
Most cloning attempts are still unsuccessful
Many clones have defects
Clones may vary in their phenotype
“noise” in gene expression
Raser and O’Shea (2005) Noise in Gene Expression:
Origins, Consequences, and Control. Science. 309
(5743): 2010-2013
Cowan, Atienza, Melton, Eggan (2005) Nuclear
Reprogramming of Somatic Cells After Fusion with Human
Embryonic Stem Cells. Science. 309 (5739):1369-1373
Using embryonic stem cells INSTEAD of egg cells
The technique is not perfect
Isolate
embryonic
stem cells
Isolate
mature
cells
Add
a marker
Add
a marker
Objectives
Describe transcription and translation
Purpose of each
Location of each
Steps of each
Proteins, enzymes, and RNA involved
Describe the roles of three types of RNA
Compare and contrast DNA replication and RNA
transcription
Use the genetic code to translate DNA into
protein
Central Dogma
Making RNA
Making Protein
Overview
transcription
translation
mRNA rRNA tRNA
mature mRNA
transcripts
ribosomal
subunits
mature
tRNA
DNARNA
•transcription
•in nucleus
RNAprotein
•translation
•in cytoplasm
•in rough ER
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Types of
RNA
transcription
translation
mRNA rRNA tRNA
mature mRNA
transcripts
ribosomal
subunits
mature
tRNA
mRNA
•messenger
•Instructions for
building a protein
rRNA
•ribosomal
•part of protein
building machinery
tRNA
•transfer
•delivers the correct
amino acids to the
ribosome
Gene Transcription
transcribed DNA
winds up again
DNA to be
transcribed unwinds
mRNA
transcript
RNA polymerase
Adding Nucleotides
growing RNA transcript
5’
3’
5’
3’
direction of transcription
DNA
A Nucleotide Subunit of RNA
phosphate
group
ribose
(sugar)
uracil (base)
Base Pairing during Transcription
DNA
DNA DNA
RNA G C A T
C G T A
G C A U
C G T A
base pairing in DNA replication base pairing in transcription
DNA duplication vs. RNA transcription
Whole strand of DNA
duplicated
Nucleotides added in one
direction only
DNA polymerase
A, T, G, C
A=T, C=G
Double helix
Short segment of DNA
transcribed
Nucleotides added in one
direction only
RNA polymerase
A, U, G, C
A=>U, C=G, T=>A
Single strand
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Transcript Modification
unit of transcription in a DNA strand
exon intron
mature mRNA transcript
poly-A
tail
5’
5’ 3’
3’
snipped
out
snipped
out
exon exon intron
cap
transcription into pre-mRNA
3’ 5’
RNA
Protein
transcription
translation
mRNA rRNA tRNA
mature mRNA
transcripts
ribosomal
subunits
mature
tRNA
•mRNA read by the
ribosome
•tRNA brings amino
acids to the ribosome
•Amino acids are
combined to make
proteins
•How many levels of
folding might this
protein go through?
•3 stages of translation
•Initiation
•Elongation
•Termination
Genetic Code
Set of 64 base triplets
Codons
61 specify amino
acids
3 stop translation
tRNA Structure
codon in mRNA
anticodon
amino
acid
OH
amino-acid
attachment site
Initiation: the pieces come
together
Initiator tRNA binds to small
ribosomal subunit
Small subunit/tRNA complex
attaches to mRNA and moves
along it to an AUG “start” codon
Large ribosomal subunit joins
complex
Elongation: the protein is made
mRNA passes through ribosomal subunits
tRNAs deliver amino acids to the ribosomal
binding site in the order specified by the mRNA
Peptide bonds form between the amino acids
and the polypeptide chain grows
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Elongation: the protein is made
Termination: all done
A stop codon moves into
place
No tRNA with anticodon
Release factors bind to the
ribosome
mRNA and polypeptide are
released
new
polypeptide
chain
mRNA
Quick Quiz Question
Some mRNA has the following sequence:
AUGCCAAAUUAUUAA
What sequence of amino acids does it code for?
What was the sequence of the DNA from which it was
transcribed?
What are some other mRNA sequences that would
produce the same protein?
Objectives
Describe three types of mutations and their
effects
What causes mutations?
How is the fate of a mutation in a somatic cell
different from that of one in a gamete?
Why is gene expression controlled?
How is gene expression controlled?
In prokaryotes
In eukaryotes
Gene Mutations
Frameshift Mutations
deletions
insertions
Base-pair substitution, a.k.a., point mutation
or single nucleotide polymorphism (SNP)
silent mutation(no effect)
nonsense mutation (stops transcription)
missense mutation (makes a different protein)
Check out these mutations:
AUGCCGCAGTAT
AUCCCGCAGTAT
AUGCCGCAGTAT
AUGCGCAGTAT
Which types of mutation are shown?
Does the gene mutation change the protein product?
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Mutations: Cause and Fix
Causes of Mutations:
Chance
Ionizing radiation, i.e., X-rays
Nonionizing radiation, i.e., UV rays
Chemicals (natural and man-made), i.e.,
cigarette smoke
Viruses, i.e., HPV
Proofreading enzymes fix mutations
Unchecked Mutations
In somatic (body) cells:
May be helpful
May be harmful
May be neither
Will NOT be passed on
In a gamete (sex cell) or asexually reproducing
organism:
May be helpful
May be harmful
May be neither
Will be passed on to offspring
This has consequences for natural selection and evolution
Why Control Gene Expression?
Making a protein is expensive:
Energy
Resources
You may not need the protein:
What is in your environment, e.g., lactose?
Eye proteins in your ear?
Blood proteins in your tongue?
Both Prokayrotes and Eukaryotes
Regulatory proteins
Negative control: stop or slow a gene’s action
Positive control: start or enhance a gene’s action
Non-coding DNA sequences
Promoter: this is where the gene starts
Enhancer: place for regulatory proteins to bind
Promoter : beginning of the gene
Operator : the repressor can bind here
Regulator : codes for the repressor
Repressor: a protein that stops transcription
Inducer : removes the repressor and allows the RNA
polymerase to act
Structural Genes : the genes for the protein
Prokaryotic Expression: the Operon
Gene Expression in Eukaryotes
You do not express all of your genes in every cell
Some of your genes, like the gene for ATP synthase, are
expressed in nearly every cell
Other genes code for proteins that only certain cells
need at certain times
This is called selective gene expression
It is responsible for cell differentiation: the process by
which different types of cells derive from one fertilized
egg
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Transcriptional: does the gene get transcribed? How
many times?
Post-transcriptional: does the RNA get processed
properly?
Translational: does the RNA get translated? How many
time?
Post-translational: does the protein get folded properly?
Eukaryotic Expression:
4 levels